EP3868776A1 - Mhc class i epitope delivering polypeptides - Google Patents

Mhc class i epitope delivering polypeptides Download PDF

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Publication number
EP3868776A1
EP3868776A1 EP21155849.9A EP21155849A EP3868776A1 EP 3868776 A1 EP3868776 A1 EP 3868776A1 EP 21155849 A EP21155849 A EP 21155849A EP 3868776 A1 EP3868776 A1 EP 3868776A1
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Prior art keywords
cell
seq
polypeptide
amino acid
present
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German (de)
English (en)
French (fr)
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Eric POMA
Erin WILLERT
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Molecular Templates Inc
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Molecular Templates Inc
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Definitions

  • peptides transported from the cytosol into the lumen of the ER by TAP are then available to be bound by different MHC class I molecules.
  • a multicomponent peptide loading machine which involves TAPs, helps assemble stable peptide-MHC class I molecule complexes and further process peptides in some instances, especially by cleavage into optimal sized peptides in a process called trimming ( see Mayerhofer P, Tamoutheastern R, J Mol Biol pii S0022-2835 (2014 )).
  • the MHC class I presentation pathway could be exploited by various therapeutics in order to induce desired immune responses; however, there are several barriers to developing such a technology, including, e.g., delivery through the cell plasma membrane; escaping the endocytotic pathway and destruction in the lysosome; and generally avoiding the sequestration, modification, and/or destruction of foreign polypeptides by the targeted cell ( Sahay G et al., J Control Release 145: 182-195 (2010 ); Fuchs H et al., Antibodies 2: 209-35 (2013 )).
  • polypeptide-containing therapeutics it is often desirable to attempt to minimize the immunogenicity of the therapeutic to prevent and/or reduce the occurrence of undesired immune responses in subjects undergoing therapeutic treatment.
  • polypeptide regions in therapeutics likely to produce B-cell and/or T-cell antigenicity and/or immunogenicity are targeted for removal, suppression, and minimization.
  • a polypeptide of the present invention comprises an embedded or inserted heterologous T-cell epitope, wherein the polypeptide is capable of intracellular delivery of the T-cell epitope from an early endosomal compartment to a proteasome of a cell in which the polypeptide is present.
  • the polypeptide of the present invention further comprises a toxin-derived polypeptide capable of routing to a subcellular compartment of a cell in which the toxin-derived polypeptide is present selected from the group consisting of: cytosol, endoplasmic reticulum, and lysosome.
  • the polypeptide of the present invention comprises a heterologous T-cell epitope is embedded or inserted in a toxin-derived polypeptide.
  • a polypeptide of the present invention comprises a toxin-derived polypeptide comprising a toxin effector polypeptide capable of exhibiting one or more toxin effector functions.
  • the toxin effector polypeptide is derived from a toxin selected from the group consisting of: ABx toxin, ribosome inactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin, bryodin, cholix toxin, claudin, diphtheria toxin, gelonin, heat-labile enterotoxin, mitogillin, pertussis toxin, pokeweed antiviral protein, pulchellin, Pseudomonas exotoxin A, restrictocin, ricin, saporin, sarcin, Shiga toxin, and subtilase cytotoxin.
  • the embedding or inserting step results in a toxin effector polypeptide capable of exhibiting one or more toxin effector functions in addition to intracellular delivery of a T-cell epitope from an early endosomal compartment to a MHC class I molecule of a cell in which the toxin effector polypeptide is present.
  • the polypeptide comprises a toxin effector polypeptide capable of intracellular delivery of a T-cell epitope from an early endosomal compartment to a proteasome of a cell in which the toxin effector polypeptide is present, and the method comprises embedding or inserting the heterologous T-cell epitope in the toxin effector polypeptide.
  • the method of creating a CD8+ T-cell epitope delivery molecule capable when present in a cell of delivering a T-cell epitope for presentation by a MHC class I molecule comprising the step of: embedding or inserting a heterologous CD8+ T-cell epitope in a proteasome delivering effector polypeptide capable of intracellular delivery of a T-cell epitope from an early endosomal compartment to a MHC class I molecule of a cell in which the proteasome delivering effector polypeptide is present.
  • the polypeptide of the present invention comprises the diphtheria toxin effector polypeptide derived from amino acids 2 to 389 of SEQ ID NO:45.
  • the toxin effector polypeptide is a Shiga toxin effector polypeptide comprising an amino acid sequence derived from an A Subunit of at least one member of the Shiga toxin family, wherein the Shiga toxin effector polypeptide comprises a disruption of at least one B-cell epitope and/or CD4+ T-cell epitope region of the Shiga toxin A Subunit amino acid sequence selected from the group of natively positioned amino acids consisting of: the B-cell epitope regions 1-15 of SEQ ID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO:1 or SEQ ID NO:2; 42-
  • a de-immunized polypeptide of the present invention comprises a heterologous CD8+ T-cell epitope disrupting an endogenous B-cell epitope and/or CD4+ T-cell epitope, wherein the polypeptide is capable of intracellular delivery of the CD8+ T-cell epitope to a MHC class I molecule from an early endosomal compartment of a cell in which the polypeptide is present.
  • the polypeptide of the present invention comprises a toxin-derived polypeptide.
  • the heterologous CD8+ T-cell epitope is in the toxin-derived polypeptide.
  • the toxin-derived polypeptide of the present invention comprises a toxin effector polypeptide.
  • the heterologous CD8+ T-cell epitope in the toxin effector polypeptide is capable of exhibiting one or more toxin effector functions.
  • the polypeptide of the present invention comprises the toxin effector polypeptide derived from a toxin selected from the group consisting of: ABx toxin, ribosome inactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin, bryodin, cholix toxin, claudin, diphtheria toxin, gelonin, heat-labile enterotoxin, mitogillin, pertussis toxin, pokeweed antiviral protein, pulchellin, Pseudomonas exotoxin A, restrictocin, ricin, saporin, sarcin, Shiga toxin, and subtilase cytotoxin.
  • a toxin selected from the group consisting of: ABx toxin, ribosome inactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin, bryodin, cholix toxin, c
  • the polypeptide of the present invention comprises the Shiga toxin effector polypeptide derived from amino acids 75 to 251 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3,. In certain further embodiments, the polypeptide of the present invention comprises the Shiga toxin effector polypeptide derived from amino acids 1 to 241 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments, the Shiga toxin effector polypeptide is derived from amino acids 1 to 251 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments, the Shiga toxin effector polypeptide is derived from amino acids 1 to 261 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
  • the toxin-derived polypeptide of the present invention comprises a toxin effector polypeptide.
  • the heterologous CD8+ T-cell epitope in the toxin effector polypeptide is capable of exhibiting one or more toxin effector functions.
  • the toxin effector polypeptide is a diphtheria toxin effector polypeptide comprising an amino acid sequence derived from the A and B Subunits of at least one member of the diphtheria toxin family, wherein the diphtheria toxin effector polypeptide comprises a disruption of at least one B-cell epitope and/or CD4+ T-cell epitope region of the amino acid sequence selected from the group of natively positioned amino acids consisting of: 3-10 of SEQ ID NO:39, 33-43 of SEQ ID NO:39, 71-77 of SEQ ID NO:39, 125-131 of SEQ ID NO:39, 138-146 of SEQ ID NO:39, 165-175 of SEQ ID NO:39, and 185-191 of SEQ ID NO:39; and wherein the diphtheria toxin effector polypeptide is capable of routing to a cytosol compartment of a cell in which the diphtheria toxin effector polypeptide is present.
  • a de-immunized polypeptide of the present invention comprises a proteasome delivering effector polypeptide comprising a first heterologous T-cell epitope disrupting an endogenous B-cell epitope and/or CD4+ T-cell epitope, wherein the proteasome delivering effector polypeptide is linked to a second CD8+ T-cell epitope; and the polypeptide is capable of intracellular delivery of the second CD8+ T-cell epitope for presentation by a MHC class I molecule on the surface of a cell in which the polypeptide is present.
  • the polypeptide of the present invention comprises a toxin-derived polypeptide.
  • the polypeptide of the present invention comprises the toxin effector polypeptide derived from a toxin selected from the group consisting of: ABx toxin, ribosome inactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin, bryodin, cholix toxin, claudin, diphtheria toxin, gelonin, heat-labile enterotoxin, mitogillin, pertussis toxin, pokeweed antiviral protein, pulchellin, Pseudomonas exotoxin A, restrictocin, ricin, saporin, sarcin, Shiga toxin, and subtilase cytotoxin.
  • a toxin selected from the group consisting of: ABx toxin, ribosome inactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin, bryodin, cholix toxin, c
  • the method comprises the steps of: identifying a CD4+ T-cell epitope in a polypeptide; and disrupting the identified CD4+ T-cell epitope with one or more amino acid residue(s) in a CD8+ T-cell epitope added to the polypeptide.
  • the disrupting step further comprises the step or steps of making one or more amino acid substitutions in the CD4+ T-cell epitope.
  • the disrupting step further comprises the step or steps of making one or more amino acid insertions in the CD4+ T-cell epitope.
  • the binding region comprises a polypeptide selected from the group consisting of: a complementary determining region 3 (CDR3) fragment constrained FR3-CDR3-FR4 (FR3-CDR3-FR4) polypeptide, single-domain antibody fragment (sdAb), nanobody, heavy-chain antibody domain derived from a camelid (V H H fragment), heavy-chain antibody domain derived from a cartilaginous fish, immunoglobulin new antigen receptors (IgNARs), V NAR fragment, single-chain variable fragment (scFv), antibody variable fragment (Fv), antigen-binding fragment (Fab), Fd fragment, small modular immunopharmaceutical (SMIP) domain, fibronection-derived 10 th fibronectin type III domain (10Fn3) (e.g.
  • the cell-targeted molecule of the present invention whereby upon administration of the cell-targeted molecule to a cell physically coupled with an extracellular target biomolecule of the binding region, the cell-targeted molecule is capable of causing death of the cell.
  • the cell-targeted molecule of the present invention whereby upon administration of the cell-targeted molecule to a first populations of cells whose members are physically coupled to extracellular target biomolecules of the binding region, and a second population of cells whose members are not physically coupled to any extracellular target biomolecule of said binding region, the cytotoxic effect of the cell-targeted molecule to members of said first population of cells relative to members of said second population of cells is at least 3-fold greater.
  • the binding region is capable of binding to an extracellular target biomolecule selected from the group consisting of: CD20, CD22, CD40, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR, EpCAM, EphB2, prostate-specific membrane antigen, Cripto, endoglin, fibroblast activated protein, Lewis-Y, CD19, CD21, CS1/ SLAMF7, CD33, CD52, EpCAM, CEA, gpA33, Mucins, TAG-72, carbonic anhydrase IX, folate binding protein, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside Lewis-Y2, VEGFR, Alpha Vbeta3, Alpha5betal, ErbB1/EGFR, Erb3, c-MET, IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANKL, FAP, Ten
  • the cell-targeted molecules of the invention comprise a Shiga toxin effector region which further comprises a mutation relative to a naturally occurring A Subunit of a member of the Shiga toxin family that changes the enzymatic activity of the Shiga toxin effector region, the mutation selected from at least one amino acid residue deletion or substitution, such as, e.g., A231E, R75A, Y77S, Y114S, E167D, R170A, R176K and/or W203A in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3
  • the cancer to be treated is selected from the group consisting of: bone cancer, breast cancer, central/peripheral nervous system cancer, gastrointestinal cancer, germ cell cancer, glandular cancer, head-neck cancer, hematological cancer, kidney-urinary tract cancer, liver cancer, lung/pleura cancer, prostate cancer, sarcoma, skin cancer, and uterine cancer.
  • the immune disorder to be treated is an immune disorder associated with a disease selected from the group consisting of: amyloidosis, ankylosing spondylitis, asthma, Crohn's disease, diabetes, graft rejection, graft-versus-host disease, Hashimoto's thyroiditis, hemolytic uremic syndrome, HIV-related diseases, lupus erythematosus, multiple sclerosis, polyarteritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, septic shock, Sjorgren's syndrome, ulcerative colitis, and vasculitis.
  • a disease selected from the group consisting of: amyloidosis, ankylosing spondylitis, asthma, Crohn's disease, diabetes, graft rejection, graft-versus-host disease, Hashimoto's thyroiditis, hemolytic uremic syndrome, HIV-related diseases, lupus erythematosus, multiple sclerosis
  • compositions comprising a polypeptide of the invention, a polypeptide and/or cell-targeted molecule comprising it, or a composition comprising any of the foregoing, for the treatment or prevention of a cancer, tumor, immune disorder, or microbial infection.
  • a composition of matter of the invention in the manufacture of a medicament for the treatment or prevention of a cancer, tumor, immune disorder, or microbial infection.
  • the present invention further provides a method for delivering exogenous material to the inside of a cell(s) in a patient in need thereof, the method comprising the step of administering to the patient a cell-targeted molecule of the present invention, wherein the target cell(s) is physically coupled with an extracellular target biomolecule of the protein of the present invention.
  • composition e.g. a pharmaceutical composition
  • diagnosis, prognosis, or characterization of a disease, disorder, or condition is the use of a compound (e.g. a polypeptide or a cell-targeted molecule) of the invention and/or composition (e.g. a pharmaceutical composition) of the invention in the diagnosis, prognosis, or characterization of a disease, disorder, or condition.
  • a compound e.g. a polypeptide or a cell-targeted molecule
  • composition e.g. a pharmaceutical composition
  • a diagnostic composition of the invention may be used to detect a cell in vivo by administering to a mammalian subject a composition comprising protein of the present invention which comprises a detection promoting agent and then detecting the presence of the protein of the present invention either in vitro or in vivo.
  • the information collected may regard the presence of a cell physically coupled with an extracellular target of the binding region of the cell-targeted molecule of the present invention and may be useful in the diagnosis, prognosis, characterization, and/or treatment of a disease, disorder, or condition.
  • Certain compounds (e.g. polypeptides and cell-targeted molecules) of the invention, compositions (e.g. pharmaceutical compositions and diagnostic compositions) of the invention, and methods of the invention may be used to determine if a patient belongs to a group that responds to a pharmaceutical composition of the invention.
  • the methods of the invention for "seeding" a tissue locus comprises administering to the chordate the cell-targeted molecule of the invention, the pharmaceutical composition of the invention, or the diagnostic composition of the invention comprising the heterologous T-cell epitope selected from the group consisting of: peptides not natively presented by the target cells of the cell-targeted molecule in MHC class I complexes, peptides not natively present within any protein expressed by the target cell, peptides not natively present within the proteome of the target cell, peptides not natively present in the extracellular microenvironment of the site to be seeded, and peptides not natively present in the tumor mass or infected tissue site to be targeted.
  • selective cytotoxicity with regard to the cytotoxic activity of a cytotoxic protein refers to the relative levels of cytotoxicity between a targeted cell population and a non-targeted bystander cell population, which can be expressed as a ratio of the half-maximal cytotoxic concentration (CD 50 ) for a targeted cell type over the CD 50 for an untargeted cell type to show preferentiality of cell killing of the targeted cell type.
  • CD 50 half-maximal cytotoxic concentration
  • lysosomal proteolysis including phagolysosome proteolysis
  • cross-presentation a process called cross-presentation, which may have evolved from a canonical ERAD system ( Gagnon E et al., Cell 110: 119-31 (2002 )).
  • certain polypeptides and proteins known or discovered to localize to lysosomes may be suitable sources for polypeptides with proteasome delivery effector regions which exhibit a proteasome delivery function(s).
  • Ribotoxic toxin effector polypeptides may be derived from the catalytic domains of members of the Ribosome Inactivating Protein (RIP) Superfamily of protein ribotoxins (de Virgilio M et al., Toxins 2: 2699-737 (2011 ); Lapadula W et al., PLoS ONE 8: e72825 (2013 ); Walsh M, Virulence 4: 774-84 (2013 )).
  • RIP Ribosome Inactivating Protein
  • the SRL is the largest universally conserved ribosomal sequence which forms a conserved secondary structure vital to the ribosome function of translocation via the cooperation of elongation factors, such as EF-Tu, EF-G, EF1, and EF2 ( Voorhees R et al., Science 330: 835-8 (2010 ); Shi X et al., J Mol Biol 419: 125-38 (2012 ); Chen K et al., PLoS One 8: e66446 (2013 )).
  • the SRL (sarcin-ricin loop) was named for being the shared target of the fungal ribotoxin sarcin and the plant type II RIP ricin.
  • the RIP Superfamily includes RIPs, fungal ribotoxins, and bacterial ribotoxins that interfere with ribosome translocation functions (see Table B; Brigotti M et al., Biochem J 257: 723-7 (1989 )).
  • Most RIPs like abrin, gelonin, ricin, and saporin, irreversibly depurinate a specific adenine in the universally conserved sarcin/ricin loop (SRL) of the large rRNAs of ribosomes (e.g. A4324 in animals, A3027 in fungi, and A2660 in prokaryotes).
  • SRL universally conserved sarcin/ricin loop
  • Most fungal ribotoxins like ⁇ -sarcin, irreversibly cleave a specific bond in the SRL (e.g. the bond between G4325 and A4326 in animals, G3028 and A3029 in fungi, and G2661 and A2662 in prokaryotes) to catalytically inhibit protein synthesis by damaging ribosomes ( Martinez-Ruiz A et al., Toxicon 37: 1549-63 (1999 ); Lacadena J et al., FEMS Microbiol Rev 31: 212-37 (2007 ); Tan Q et al., J Biotechnol 139: 156-62 (2009 )).
  • the bacterial protein ribotoxins Ct, DT, and PE are classified in the RIP Superfamily because they can inhibit protein synthesis by catalytically damaging ribosome function and induce apoptosis efficiently with only a few toxin molecules.
  • Type I RIPs e.g. gelonin, luffins, PAP, saporins and trichosanthins
  • Type II RIPs e.g . abrin, ricin, Shiga toxins
  • Type III RIPs e.g.
  • barley JIP60 RIP and maize b-32 RIP are synthesized as proenzymes that require extensive proteolytic processing for activation ( Peumans W et al., FASEB J 15: 1493-1506 (2001 ); Mak A et al., Nucleic Acids Res 35: 6259-67 (2007 )).
  • potently cytotoxic immunotoxins have been generated using polypeptides derived from the RIPs: ricin, gelonin, saporin, momordin, and PAPs ( Pasqualucci L et al., Haematologica 80: 546-56 (1995 )).
  • cytosol targeting toxin effector polypeptide refers to a polypeptide derived from proteins, including naturally occurring ribotoxins and synthetic ribotoxins, which are capable of routing intracellularly to the cytosol after cellular internalization.
  • cytosolic targeting toxin effector regions are derived from naturally occurring protein toxins or toxin-like structures which are altered or engineered by human intervention, however, other polypeptides, such as, e.g., computational designed polypeptides, are within the scope of the term as used herein (see e.g.
  • Non-limiting examples of non-toxin derived molecules with endosomal escape functions include: viral agents like hemagglutinin HA2; vertebrate derived polypeptides and peptides like human calcitonin derived peptides, bovine prion protein, and sweet arrow peptide; synthetic biomimetic peptides; and polymers with endosome disrupting abilities (see e.g. Varkouhi A et al., J Control Release 151: 220-8 (2010 )).
  • Escape from endosomal compartments, including lysosomes, can be measured directly and quantitated using assays known in the art, such as, e.g., using reporter assays with horseradish peroxidase, bovine serum albumin, fluorophores like Alexa 488, and toxin derived polypetides (see e.g. Bartz R et al., Biochem J 435: 475-87 (2011 ); Gilabert-Oriol, R et al., Toxins 6: 1644-66 (2014 )).
  • assays known in the art such as, e.g., using reporter assays with horseradish peroxidase, bovine serum albumin, fluorophores like Alexa 488, and toxin derived polypetides (see e.g. Bartz R et al., Biochem J 435: 475-87 (2011 ); Gilabert-Oriol, R et al., Toxins 6: 1644-66
  • polypeptides comprising an endoplasmic retention/retrieval signal motif (e.g. KDEL) can localize to the ER of a eukaryotic cell from different compartments within the cell.
  • endoplasmic retention/retrieval signal motif e.g. KDEL
  • a proteasome delivery effector polypeptide Once a proteasome delivery effector polypeptide is obtained, it can be engineered into a T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide of the present invention using the methods of the present invention.
  • one or more T-cell epitopes are embedded, fused, or inserted into any proteasome delivery effector polypeptide, such as, e.g., a toxin effector polypeptide which routes to the cytosol (which may include a ribotoxic toxin effector polypeptide), in order to create polypeptides of the present invention, which starting from an early endosomal compartment are capable of delivering a T-cell epitope to the proteasome for entry into the MHC class I pathway and subsequent MHC class I presentation.
  • a proteasome delivery effector polypeptide such as, e.g., a toxin effector polypeptide which routes to the cytosol (which may include a ribotoxic toxin effector polypeptide)
  • a given molecule's ability to deliver T-cell epitopes to the proteasome for entry into the MHC class I pathway of a cell may be assayed by the skilled worker using the methods described herein and/or techniques known in the art (see Examples, infra).
  • a given molecule's ability to deliver a T-cell epitope from an early endosome compartment to a proteasome may be assayed by the skilled worker using the methods described herein and/or techniques known in the art.
  • a given molecule's ability to deliver a T-cell epitope from an early endosome compartment to a MHC class I molecule for presention on the surface of a cell may be assayed by the skilled worker using the methods described herein and/or techniques known in the art (see Examples, infra ).
  • a given molecule's ability to deliver a T-cell epitope from an early endosome compartment to a MHC class I molecule may be assayed by the skilled worker using the methods described herein and/or techniques known in the art.
  • the polypeptides and cell-targeted molecules of the present invention each comprise one or more heterologous T-cell epitopes.
  • a T-cell epitope is a molecular structure which is comprised by an antigen and can be represented by a peptide or linear amino acid sequence and.
  • a heterologous T-cell epitope is an epitope not already present in the source polypeptide are starting proteasome delivery effector polypeptide that is modified using a method of the present invention in order to create a T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide of the present invention.
  • the heterologous T-cell epitope peptide may be incorporated into the source polypeptide via numerous methods known to the skilled worker, including, e.g., the processes of creating one or more amino acid substitutions within the source polypeptide, fusing one or more amino acids to the source polypeptide, inserting one or more amino acids into the source polypeptide, linking a peptide to the source polypeptide, and/or a combination of the aforementioned processes.
  • the result is a modified variant of the source polypeptide which comprises one or more heterologous T-cell epitopes.
  • T-cell epitopes may be derived from a number of sources, including peptide components of proteins and peptides derived from proteins already known or shown to be capable of eliciting a mammalian immune response. T-cell epitopes may be created or derived from various naturally occurring proteins. T-cell epitopes may be derived from various naturally occurring proteins foreign to mammals, such as, e.g., proteins of microorganisms. In particular, infectious microorganisms may contain numerous proteins with known antigenic and/or immunogenic properties or sub-regions or epitopes. T-cell epitopes may be derived from mutated human proteins and/or human proteins aberrantly expressed by malignant human cells.
  • T-cell epitopes may be chosen or derived from a number of source molecules already known to be capable of eliciting a mammalian immune response, including peptides, peptide components of proteins, and peptides derived from proteins.
  • the proteins of intracellular pathogens with mammalian hosts are sources for T-cell epitopes.
  • T-cell epitopes can be selected or identified from human viruses or other intracellular pathogens, such as, e.g., bacteria like mycobacterium, fungi like toxoplasmae, and protists like trypanosomes.
  • HA glycoproteins FE17 S139/1, CH65, C05
  • HA1 hemagglutin 1
  • HA2 hemagglutinin 2
  • NS1 and NS 2 nonstructural protein 1 and 2
  • M1 and M2 matrix protein 1 and 2
  • NP nucleoprotein
  • NA neuraminidase
  • T-cell epitope While any T-cell epitope may be used in the compositions and methods of the present invention, certain T-cell epitopes may be preferred based on their known and/or empirically determined characteristics.
  • the MHC gene encodes multiple MHC-I molecular variants. Because MHC class I protein polymorphisms can affect antigen-MHC class I complex recognition by CD8+ T-cells, heterologous T-cell epitopes may be chosen using based on knowledge about certain MHC class I polymorphisms and/or the ability of certain antigen-MHC class I complexes to be recognized by T-cells of different genotypes.
  • T-cell epitopes may be chosen for use as a heterologous T-cell epitope component of the present invention based on the peptide selectivity of the HLA variants encoded by the alleles more prevalent in certain human populations.
  • the human population is polymorphic for the alpha chain of MHC class I molecules, and the variable alleles are encoded by the HLA genes.
  • Certain T-cell epitopes may be more efficiently presented by a specific HLA molecule, such as, e.g., the commonly occurring HLA variants encoded by the HLA-A allele groups HLA-A2 and HLA-A3.
  • T-cell epitopes for use as a heterologous T-cell epitope component of the present invention, multiple factors in the process of epitope selection by MHC class I molecules may be considered that can influence epitope generation and transport to receptive MHC class I molecules, such as, e.g., the epitope specificity of the following factors in the target cell: proteasome, ERAAP/ERAP1, tapasin, and TAPs can (see e.g. Akram A, Inman R, Clin Immunol 143: 99-115 (2012 )).
  • proteasome delivery effector polypeptides are immunogenic in extracellular spaces when administered to vertebrates. Unwanted immunogenicity in protein therapeutics has resulted in reduced efficacy, unpredictable pharmacokinetics, and undesirable immune responses that limit dosages and repeat administrations.
  • one main challenge is silencing or disrupting immunogenic epitopes within a polypeptide effector domain, e.g . its cytosolic targeting domain, while retaining the desired polypeptide effector function(s), such as, e.g., proteasome delivery.
  • the polypeptides of the present invention may be coupled to numerous other polypeptides, agents, and moieties to create cell-targeted molecules, such as, e.g . cytotoxic, cell-targeted proteins of the present invention.
  • Cytotoxic polypeptides and proteins may be constructed using the T-cell epitope comprising proteasome delivering effector polypeptides of the invention and the addition of cell-targeting components, such as, e.g., a binding region capable of exhibiting high affinity binding to an extracellular target biomolecule physically-coupled to the surface of a specific cell type(s).
  • the B-cell epitope de-immunized polypeptides of the present invention may be used as components of numerous useful molecules for administration to mammals.
  • Binding regions of the cell-targeted molecules of the present invention comprise one or more polypeptides capable of selectively and specifically binding an extracellular target biomolecule. Binding regions may comprise one or more various polypeptide moieties, such as ligands whether synthetic or naturally occurring ligands and derivatives thereof, immunoglobulin derived domains, synthetically engineered scaffolds as alternatives to immunoglobulin domains, and the like.
  • the use of proteinaceous binding regions in cell-targeting molecules of the invention allows for the creation of cell-targeting molecules which are single-chain, cell-targeting proteins.
  • An immunoglobulin-type binding region may be a polypeptide sequence of an antibody or antigen-binding fragment thereof wherein the amino acid sequence has been varied from that of a native antibody or an Ig-like domain of a non-immunoglobulin protein, for example by molecular engineering or selection by library screening. Because of the relevance of recombinant DNA techniques and in vitro library screening in the generation of immunoglobulin-type binding regions, antibodies can be redesigned to obtain desired characteristics, such as smaller size, cell entry, or other therapeutic improvements. The possible variations are many and may range from the changing of just one amino acid to the complete redesign of, for example, a variable region. Typically, changes in the variable region will be made in order to improve the antigen-binding characteristics, improve variable region stability, or reduce the potential for immunogenic responses.
  • the binding region of the present proteins is selected from the group which includes single-domain antibody domains (sdAbs), nanobodies, heavy-chain antibody domains derived from camelids (V H H fragments), bivalent nanobodies, heavy-chain antibody domains derived from cartilaginous fishes, immunoglobulin new antigen receptors (IgNARs), V NAR fragments, single-chain variable (scFv) fragments, multimerizing scFv fragments (diabodies, triabodies, tetrabodies), bispecific tandem scFv fragments, disulfide stabilized antibody variable (Fv) fragments, disulfide stabilized antigen-binding (Fab) fragments consisting of the V L , V H , C L and C H 1 domains, divalent F(ab')2 fragments, Fd fragments consisting of
  • Certain cell-targeted molecules of the present invention comprise a polypeptide of the present invention linked to an extracellular target biomolecule specific binding region comprising one or more polypeptides capable of selectively and specifically binding an extracellular target biomolecule.
  • Extracellular target biomolecules may be selected based on numerous criteria.
  • the phrase "physically coupled" when used to describe a target biomolecule means both covalent and/or non-covalent intermolecular interactions that couple the target biomolecule, or a portion thereof, to the outside of a cell, such as a plurality of non-covalent interactions between the target biomolecule and the cell where the energy of each single interaction is on the order of about 1-5 kiloCalories (e.g. electrostatic bonds, hydrogen bonds, Van der Walls interactions, hydrophobic forces, etc.). All integral membrane proteins can be found physically coupled to a cell membrane, as well as peripheral membrane proteins.
  • nucleated vertebrate cells are believed to be capable of presenting intracellular peptide epitopes using the MHC class I system.
  • extracellular target biomolecules of the cell-targeted molecules of the invention may in principle target any nucleated vertebrate cell for T-cell epitope delivery into the MHC class I presentation pathway.
  • the carboxy-terminal lysine-asparagine-glutamate-leucine (KDEL) sequence is a canonical, endoplasmic reticulum retention and retrieval signal motif for soluble proteins in eukaryotic cells and is recognized by the KDEL receptors (see, Capitani M, Sallese M, FEBS Lett 583: 3863-71 (2009 ), for review).
  • the KDEL signal motif family includes at least 46 polypeptide variants shown using synthetic constructs ( Raykhel, J Cell Biol 179: 1193-204 (2007 )). Additional KDEL signal motifs include ALEDEL, HAEDEL, HLEDEL, KLEDEL, IRSDEL, ERSTEL, and RPSTEL ( Alanen H et al., J Mol Biol 409: 291-7 (2011 )). A generalized consensus motif representing the majority of KDEL signal motifs has been described as [KRHQSA]-[DENQ]-E-L ( Hulo N et al., Nucleic Acids Res 34: D227-30 (2006 )).
  • KDEL receptors Proteins containing KDEL family signal motifs are bound by KDEL receptors distributed throughout the Golgi complex and transported to the endoplasmic reticulum by a microtubule-dependent mechanism for release into the lumen of the endoplasmic reticulum ( Griffiths G et al., J Cell Biol 127: 1557-74 (1994 ); Miesenböck G, Rothman J, J Cell Biol 129: 309-19 (1995 )). KDEL receptors dynamically cycle between the Golgi complex and endoplasmic reticulum ( Jackson M et al., EMBO J. 9: 3153-62 (1990 ); Schutze M et al., EMBO J. 13: 1696-1705 (1994 )).
  • the general structure of the cell-targeted molecules of the present invention is modular, in that various, diverse cell-targeting binding regions may be used with various CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides to provide for diverse targeting of various extracellular target biomolecules and thus targeting of cytotoxicity, cytostasis, and/or exogenous material delivery to various diverse cell types.
  • Individual cell-targeting moiety, polypeptide, and/or protein components of the present invention may be suitably linked to each other via one or more linkers well known in the art and/or described herein.
  • Individual polypeptide subcomponents of the binding regions e.g . heavy chain variable regions (V H ), light chain variable regions (V L ), CDR, and/or ABR regions, may be suitably linked to each other via one or more linkers well known in the art and/or described herein (see e.g.
  • Suitable linkers may be proteinaceous and comprise one or more amino acids, peptides, and/or polypeptides. Proteinaceous linkers are suitable for both recombinant fusion proteins and chemically linked conjugates.
  • a proteinaceous linker typically has from about 2 to about 50 amino acid residues, such as, e.g., from about 5 to about 30 or from about 6 to about 25 amino acid residues. The length of the linker selected will depend upon a variety of factors, such as, e.g., the desired property or properties for which the linker is being selected (see e.g. Chen X et al., Adv Drug Deliv Rev 65: 1357-69 (2013 )).
  • non-proteinaceous chemical linkers include but are not limited to N-succinimidyl (4-iodoacetyl)-aminobenzoate, S- ( N -succinimidyl) thioacetate (SATA), N-succinimidyl-oxycarbonyl-cu-methyl-a-(2-pyridyldithio) toluene (SMPT), N-succinimidyl 4-(2-pyridyldithio)-pentanoate (SPP), succinimidyl 4-(N-maleimidomethyl) cyclohexane carboxylate (SMCC or MCC), sulfosuccinimidyl (4-iodoacetyl)-aminobenzoate, 4-succinimidyl (4-iodoacetyl)-aminobenzoate, 4-succinimidyl (4-iodoacetyl)-aminobenzo
  • Proteinaceous linkers may be chosen for incorporation into recombinant fusion cell-targeted molecules of the present invention.
  • linkers typically comprise about 2 to 50 amino acid residues, preferably about 5 to 30 amino acid residues ( Argos P, J Mol Biol 211: 943-58 (1990 ); Williamson M, Biochem J 297: 240-60 (1994 ); George R, Heringa J, Protein Eng 15: 871-9 (2002 ); Kreitman R, AAPS J 8: E532-51 (2006 )).
  • proteinaceous linkers comprise a majority of amino acid residues with polar, uncharged, and/or charged residues, such as, e.g., threonine, proline, glutamine, glycine, and alanine (see e.g. Huston J et al. Proc Natl Acad Sci U.S.A. 85: 5879-83 (1988 ); Pastan I et al., Annu Rev Med 58: 221-37 (2007 ); Li J et al., Cell Immunol 118: 85-99 (1989 ); Cumber A et al.
  • polar, uncharged, and/or charged residues such as, e.g., threonine, proline, glutamine, glycine, and alanine
  • the retention of "significant" Shiga toxin effector function refers to a level of Shiga toxin functional activity, as measured by an appropriate quantitative assay with reproducibility comparable to a wild-type Shiga toxin effector polypeptide control.
  • significant Shiga toxin effector function is exhibiting an IC 50 of 300 pM or less depending on the source of the ribosomes ( e.g. bacteria, archaea, or eukaryote (algae, fungi, plants, or animals)).
  • a Shiga toxin effector polypeptide of the present invention may comprise or consist essentially of a full-length or truncated Shiga toxin A Subunit with at least one mutation, e.g . deletion, insertion, inversion, or substitution, in a provided B-cell and/or CD4+ T-cell epitope region.
  • the polypeptides comprise a disruption which comprises a deletion of at least one amino acid within the B-cell and/or CD4+ T-cell epitope region.
  • the polypeptides comprise a disruption which comprises an insertion of at least one amino acid within the B-cell and/or CD4+ T-cell epitope region.
  • Assays for diphtheria toxin effector activity can measure various activities, such as, e.g., protein synthesis inhibitory activity, ADP-ribosylation, inhibition of cell growth, and/or cytotoxicity. Sufficient subcellular routing may be merely deduced by observing cytotoxicity in cytotoxicity assays, such as, e.g., cytotoxicity assays based on T-cell epitope presentation or based on a toxin effector function involving a cytosolic and/or ER target substrate.
  • cytotoxicity assays such as, e.g., cytotoxicity assays based on T-cell epitope presentation or based on a toxin effector function involving a cytosolic and/or ER target substrate.
  • Such modifications are well known to the skilled worker and include, for example, a methionine added at the amino terminus to provide an initiation site, additional amino acids placed on either terminus to create conveniently located restriction sites or termination codons, and biochemical affinity tags fused to either terminus to provide for convenient detection and/or purification.
  • luffins there are several amino acid residues important for catalytic activity, such as, e.g., tyrosine-71, glutamate-86, tyrosine-111, glutamate-160, and arginine-163 ( Ma Q et al., Acta Crystallogr D Biol Crystallogr 56: 185-6 (2000 ))
  • the cytotoxicity of the A Subunits of members of the Shiga toxin family may be altered, reduced, or eliminated by mutation or truncation.
  • the positions labeled tyrosine-77, glutamate-167, arginine-170, tyrosine-114, and tryptophan-203 have been shown to be important for the catalytic activity of Stx, Stx1, and Stx2 ( Hovde C et al., Proc Natl Acad Sci USA 85: 2568-72 (1988 ); Deresiewicz R et al., Biochemistry 31: 3272-80 (1992 ); Deresiewicz R et al., Mol Gen Genet 241: 467-73 (1993 ); Ohmura M et al., Microb Pathog 15: 169-76 (1993 ); Cao C et al., Microbiol Immunol 38: 441-7 (1994 ); Suhan M, Hovde C, Infect Immun 66: 5252-9 (19
  • CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides which may be used as components of various compositions of matter, such as cell-targeted cytotoxic molecules and diagnostic compositions.
  • CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides have uses as components of various protein therapeutics, such as, e.g. immunotoxins and ligand-toxin fusions, for the targeted killing of specific cell types for the treatment of a variety of diseases, including cancers, immune disorders, and microbial infections.
  • any CD8+ T-cell hyper-immunized, polypeptide of the invention may be engineered into a potentially useful, therapeutic, cell-targeted molecule with the addition of a cell-targeting moiety which targets cellular internalization to a specific cell-type(s) within a chordate, such as, e.g., an amphibian, bird, fish, mammal, reptile, or shark.
  • a cell-targeting moiety which targets cellular internalization to a specific cell-type(s) within a chordate
  • any B-cell epitope de-immunized polypeptide of the invention may be engineered into a potentially useful, therapeutic, cell-targeted molecule with the addition of a cell-targeting moiety which targets cellular internalization to a specific cell-type(s) within a chordate.
  • the present invention provides various cytotoxic cell-targeted molecules comprising CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides functionally associated with binding regions to effectuate cell targeting such that the cytotoxic cell-targeted molecules selectively delivery T-cell epitopes, kill, inhibit the growth of, deliver exogenous material to, and/or detect specific cell types.
  • This system is modular, in that any number of diverse binding regions may be used to target to diverse cell types any CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide of the invention, including.
  • the presentation of a T-cell immunogenic epitope peptide by the MHC class I complex targets the presenting cell for killing by CTL-mediated cytolysis.
  • MHC class I peptides into proteasome delivering effector polypeptide components of target-cell-internalizing therapeutics
  • the targeted delivery and presentation of immuno-stimulatory antigens may be accomplished by harnessing vertebrate target cells' endogenous MHC class I pathways.
  • the presentation by targeted cells of immuno-stimulatory non-self antigens such as, e.g., known viral epitope-peptides with high immunogenicity, can signal to other immune cells to destroy the target cells and recruit more immune cells to the target cell site within an organism.
  • parental cytotoxic molecules which rely on toxin and/or enzymatic regions for cytotoxicity may be engineered to be cytotoxic only via T-cell epitope cytosolic delivery and presentation by embedding a T-cell epitope in the enzymatic domain of the parental molecule such that the enzymatic activity is reduced or eliminated by the sequence changes that create the heterologous T-cell epitope.
  • This allows for the one-step modification of enzymatically-cytotoxic molecules, which have the ability to internalize into cells and route to the cytosol, into enzymatically inactive, cytotoxic molecules which rely on T-cell epitope proteasome delivery and presentation for cytotoxicity and local immuno-stimulation.
  • Certain embodiments of the CD8+ T-cell hyper-immunized polypeptides and cell-targeted molecules of the present invention are capable of delivering one or more T-cell epitopes to the proteasome of a target cell.
  • the delivered T-cell epitope are then proteolytic processed and presented by the MHC class I pathway on the outside surface of the target cell.
  • T-cell epitope presenting functions of the CD8+ T-cell hyper-immunized polypeptides and cell-targeted molecules of the present invention are vast. Every nucleated cell in a mammalian organism may be capable of MHC class I pathway presentation of immunogenic T-cell epitope peptides on their cell outer surfaces complexed to MHC class I molecules. In addition, the sensitivity of T-cell epitope recognition is so seventeen that only a few MHC-I peptide complexes are required to be presented- even presentation of a single complex can be sufficient for recognition by an effector T-cell ( Sykulev Y et al., Immunity 4: 565-71 (1996 )).
  • the CD8+ T-cell hyper-immunized polypeptide must first reach the interior of a target cell and then come in contact with a proteasome in the target cell.
  • cell-targeting molecules of the present invention In order to deliver a CD8+ T-cell hyper-immunized polypeptide of the present invention to the interior of a target cell, cell-targeting molecules of the present invention must be capable of target cell internalization.
  • the CD8+ T-cell hyper-immunized polypeptide of the invention Once the CD8+ T-cell hyper-immunized polypeptide of the invention is internalized as a component of a cell-targeting molecule, the CD8+ T-cell hyper-immunized polypeptide will typical reside in an early endosomal compartment, such as, e.g., endocytotic vesicle.
  • the CD8+ T-cell hyper-immunized polypeptide then has to reach a target cell's proteasome with at least one intact, heterologous T-cell epi
  • An alternative method for direct identification and quantification of MHC I/peptide complexes involves mass spectrometry analyses, such as, e.g., the ProPresent Antigen Presentation Assay (Prolmmune, Inc., Sarasota, FL, U.S.) in which peptide-MCH class I complexes are extracted from the surfaces of cells, then the peptides are purified and identified by sequencing mass spectrometry ( Falk K et al., Nature 351: 290-6 (1991 )).
  • mass spectrometry analyses such as, e.g., the ProPresent Antigen Presentation Assay (Prolmmune, Inc., Sarasota, FL, U.S.) in which peptide-MCH class I complexes are extracted from the surfaces of cells, then the peptides are purified and identified by sequencing mass spectrometry ( Falk K et al., Nature 351: 290-6 (1991 )).
  • MHC-peptide binding assays based on a measure of the ability of a peptide to stabilize the ternary MHC-peptide complex for a given MHC Class I allele, as a comparison to known controls, have been developed (e.g., MHC-peptide binding assay from Prolmmmune, Inc.).
  • Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vitro or in vivo investigation to monitor killing of epitope specific CSFE labeled target cells ( Durward M et al., J Vis Exp 45 pii 2250 (2010 )).
  • MHC Class I/antigen promoting agent e.g., a peptide, protein or inactivated/attenuated virus vaccine
  • active agent e.g. a virus
  • CTL activity can be monitored for CTL activity with methods similar to those described previously (e.g. CTL cytotoxicity assays and quantification of cytokine release).
  • HLA-A, HLA-B, and/or HLA-C molecules are isolated from the intoxicated cells after lysis using immune affinity (e.g., an anti-MHC antibody "pulldown" purification) and the associated peptides (i.e., the peptides presented by the isolated MHC molecules) are recovered from the purified complexes.
  • the recovered peptides are analyzed by sequencing mass spectrometry.
  • the mass spectrometry data is compared against a protein database library consisting of the sequence of the exogenous (non-self) peptide (T-cell epitope X) and the international protein index for humans (representing "self” or non-immunogenic peptides).
  • the peptides are ranked by significance according to a probability database.
  • the set of presented peptide-antigen-MHC complexes can vary between cells due to the antigen-specific HLA molecules expressed. T-cells can then recognize specific peptide-antigen-MHC complexes displayed on a cell surface using different TCR molecules with different antigen-specificities.
  • Cell-targeted molecules of the present invention comprising CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides of the present invention can provide both: 1) cell type specific T-cell-epitope delivery for MHC class I presentation and 2) potent cytotoxicity.
  • certain embodiments of the cell-targeted molecules of the present invention also provide de-immunizaiton, which reduces the likelihood of certain immune responses when administered to a mammal.
  • the cell-targeted molecule of the invention upon contacting a cell physically coupled with an extracellular target biomolecule of the cell-targeting moiety (e.g. a cell-targeted binding region), the cell-targeted molecule of the invention is capable of causing death of the cell.
  • the mechanism of cell kill may be direct, e.g . via the enzymatic activity of a toxin effector region, or indirect via CTL-mediated cytolysis, and may be under varied conditions of target cells, such as an ex vivo manipulated target cell, a target cell cultured in vitro, a target cell within a tissue sample cultured in vitro, or a target cell in vivo.
  • T-cell epitope delivering, CD8+ T-cell hyper-immunized polypeptides of the present invention, with or without B-cell epitope de-immunization may be used as components of cell-targeted molecules for indirect cell kill.
  • Certain embodiments of the cell-targeted molecules of the present invention are cytotoxic because they comprise a CD8+ T-cell epitope presenting polypeptide of the invention which delivers one or more T-cell epitopes to the MHC class I presentation pathway of a target cell upon target internalization of the cell-targeted molecule.
  • the cell-targeted molecule of the present invention upon contacting a cell physically coupled with an extracellular target biomolecule of the cell-targeting moiety (e.g. a cell-targeted binding region), the cell-targeted molecule of the invention is capable of indirectly causing the death of the cell, such as, e.g., via the presentation of one or more T-cell epitopes by the target cell and the subsequent recruitment of CTLs.
  • an extracellular target biomolecule of the cell-targeting moiety e.g. a cell-targeted binding region
  • T-cell epitope delivering, CD8+ T-cell hyper-immunized, and/or B-cell/CD4+ T-cell de-immunized polypeptides of the present invention may be used as components of cell-targeted molecules for direct cell kill.
  • the cell-targeted molecule of the present invention upon contacting a cell physically coupled with an extracellular target biomolecule of the cell-targeting moiety (e .g. a cell-targeted binding region), the cell-targeted molecule of the invention is capable of directly causing the death of the cell, such as, e.g., via the enzymatic activity of a toxin effector region.
  • an extracellular target biomolecule of the cell-targeting moiety e.g. a cell-targeted binding region
  • cell-targeted molecules of the present invention optionally may be used for information gathering and diagnostic functions.
  • non-toxic variants of the cytotoxic, cell-targeted molecules of the invention, or optionally toxic variants may be used to deliver additional exogenous materials to and/or label the interiors of cells physically coupled with an extracellular target biomolecule of the cytotoxic protein.
  • Various types of cells and/or cell populations which express target biomolecules to at least one cellular surface may be targeted by the cell-targeted molecules of the invention for receiving exogenous materials.
  • the functional components of the present invention are modular, in that various toxin effector regions and additional exogenous materials may be linked to various binding regions to provide diverse applications, such as non-invasive in vivo imaging of tumor cells.
  • CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides which become non-toxic after T-cell epitope addition may still be useful for delivering exogenous materials into cells (e.g. T-cell epitope replacements overlapping amino acid resides critical for catalytic function of a toxin effector region).
  • Certain cell-targeted molecules of the present invention have uses in the in vitro and/or in vivo detection of specific cells, cell types, and/or cell populations.
  • the proteins described herein are used for both diagnosis and treatment, or for diagnosis alone.
  • the cytotoxic protein variant which incorporates a detection promoting agent for diagnosis may be rendered nontoxic by catalytic inactivation of a toxin effector region via one or more amino acid substitutions, including exemplary substitutions described herein.
  • Nontoxic forms of the cytotoxic, cell-targeted molecules of the invention that are conjugated to detection promoting agents optionally may be used for diagnostic functions, such as for companion diagnostics used in conjunction with a therapeutic regimen comprising the same or a related binding region.
  • diagnostic embodiments of the cell-targeted molecules of the invention may be used for information gathering via various imaging techniques and assays known in the art.
  • diagnostic embodiments of the cell-targeted molecules of the invention may be used for information gathering via imaging of intracellular organelles (e.g. endocytotic, Golgi, endoplasmic reticulum, and cytosolic compartments) of individual cancer cells, immune cells, or infected cells in a patient or biopsy sample.
  • intracellular organelles e.g. endocytotic, Golgi, endoplasmic reticulum, and cytosolic compartments
  • Various types of information may be gathered using the diagnostic embodiments of the cell-targeted molecules of the invention whether for diagnostic uses or other uses. This information may be useful, for example, in diagnosing neoplastic cell types, determining therapeutic susceptibilities of a patient's disease, assaying the progression of antineoplastic therapies over time, assaying the progression of immunomodulatory therapies over time, assaying the progression of antimicrobial therapies over time, evaluating the presence of infected cells in transplantation materials, evaluating the presence of unwanted cell types in transplantation materials, and/or evaluating the presence of residual tumor cells after surgical excision of a tumor mass.
  • the CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides and cell-targeted molecules of the present invention may be produced using biochemical engineering techniques well known to those of skill in the art.
  • polypeptides and cell-targeted molecules of the invention may be manufactured by standard synthetic methods, by use of recombinant expression systems, or by any other suitable method.
  • polypeptides and cell-targeted proteins of the present invention may be synthesized in a number of ways, including, e.g .
  • methods comprising: (1) synthesizing a polypeptide or polypeptide component of a protein using standard solid-phase or liquid-phase methodology, either stepwise or by fragment assembly, and isolating and purifying the final peptide compound product; (2) expressing a polynucleotide that encodes a polypeptide or polypeptide component of a cell-targeted protein of the invention in a host cell and recovering the expression product from the host cell or host cell culture; or (3) cell-free in vitro expression of a polynucleotide encoding a polypeptide or polypeptide component of a cell-targeted protein of the invention, and recovering the expression product; or by any combination of the methods of (1), (2) or (3) to obtain fragments of the peptide component, subsequently joining (e.g. ligating) the fragments to obtain the peptide component, and recovering the peptide component.
  • a CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide or a protein or polypeptide component of a cell-targeted protein of the invention may suitably be manufactured by standard synthetic methods.
  • peptides may be synthesized by, e.g . methods comprising synthesizing the peptide by standard solid-phase or liquid-phase methodology, either stepwise or by fragment assembly, and isolating and purifying the final peptide product.
  • CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides and cytotoxic, cell-targeted proteins of the present invention may be prepared (produced and purified) using recombinant techniques well known in the art.
  • methods for preparing polypeptides by culturing host cells transformed or transfected with a vector comprising the encoding polynucleotide and recovering the polypeptide from cell culture are described in, e.g.
  • Any suitable host cell may be used to produce a polypeptide and/or cell-targeted protein of the invention.
  • Host cells may be cells stably or transiently transfected, transformed, transduced or infected with one or more expression vectors which drive expression of a polypeptide of the invention.
  • a CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides and/or cell-targeted protein of the invention may be produced by modifying the polynucleotide encoding a polypeptide or cell-targeted protein of the invention that result in altering one or more amino acids or deleting or inserting one or more amino acids in order to achieve desired properties, such as changed cytotoxicity, changed cytostatic effects, and/or changed serum half-life.
  • host organisms for expression of cell-targeted proteins of the invention include prokaryotes, such as E. coli and B. subtilis, eukaryotic cells, such as yeast and filamentous fungi (like S. cerevisiae, P. pastoris, A. awamori, and K. lactis), algae (like C. reinhardtii), insect cell lines, mammalian cells (like CHO cells), plant cell lines, and eukaryotic organisms such as transgenic plants (like A. thaliana and N. benthamiana).
  • prokaryotes such as E. coli and B. subtilis
  • eukaryotic cells such as yeast and filamentous fungi (like S. cerevisiae, P. pastoris, A. awamori, and K. lactis), algae (like C. reinhardtii), insect cell lines, mammalian cells (like CHO cells), plant cell lines, and eukaryotic organisms such as transgenic plants (like A. thaliana and N. bent
  • compositions Comprising a T-Cell Hyper-Immunized and/or B-Cell/CD4+ T-Cell De-Immunized Polypeptide of the Present Invention or Cell-Targeted Molecule Comprising the Same
  • the present invention provides polypeptides and proteins for use, alone or in combination with one or more additional therapeutic agents, in a pharmaceutical composition, for treatment or prophylaxis of conditions, diseases, disorders, or symptoms described in further detail below (e.g. cancers, malignant tumors, non-malignant tumors, growth abnormalities, immune disorders, and microbial infections).
  • the present invention further provides pharmaceutical compositions comprising a polypeptide or cell-targeted molecule of the invention, or a pharmaceutically acceptable salt or solvate thereof, according to the invention, together with at least one pharmaceutically acceptable carrier, excipient, or vehicle.
  • the pharmaceutical composition of the present invention may comprise homo-multimeric and/or hetero-multimeric forms of the polypeptides or cell-targeted molecules of the invention.
  • prevention refers to an approach for preventing the development of, or altering the pathology of, a condition, disease, or disorder. Accordingly, “prevention” may refer to prophylactic or preventive measures.
  • beneficial or desired clinical results include, but are not limited to, prevention or slowing of symptoms, progression or development of a disease, whether detectable or undetectable.
  • a subject e.g. a human
  • prevention includes slowing the onset of disease relative to the absence of treatment, and is not necessarily meant to imply permanent prevention of the relevant disease, disorder or condition.
  • preventing or “prevention” of a condition may in certain contexts refer to reducing the risk of developing the condition, or preventing or delaying the development of symptoms associated with the condition.
  • Diagnostic compositions comprise a polypeptide or cell-targeted molecule of the invention and one or more detection promoting agents.
  • detection promoting agents are known in the art, such as isotopes, dyes, colorimetric agents, contrast enhancing agents, fluorescent agents, bioluminescent agents, and magnetic agents. These agents may be incorporated into the polypeptide or cell-targeted molecule of the invention at any position.
  • the incorporation of the agent may be via an amino acid residue(s) of the protein or via some type of linkage known in the art, including via linkers and/or chelators.
  • the incorporation of the agent is in such a way to enable the detection of the presence of the diagnostic composition in a screen, assay, diagnostic procedure, and/or imaging technique.
  • a cell-targeted molecule of the invention may be directly or indirectly linked to one or more detection promoting agents.
  • detection promoting agents known to the skilled worker which can be operably linked to the polypeptides or cell-targeted molecules of the invention for information gathering methods, such as for diagnostic and/or prognostic applications to diseases, disorders, or conditions of an organism (see e.g.
  • solvate in the context of the present invention refers to a complex of defined stoichiometry formed between a solute (in casu, a polypeptide compound or pharmaceutically acceptable salt thereof according to the invention) and a solvent.
  • the solvent in this connection may, for example, be water, ethanol or another pharmaceutically acceptable, typically small-molecular organic species, such as, but not limited to, acetic acid or lactic acid.
  • a solvate is normally referred to as a hydrate.
  • Polypeptides and proteins of the present invention, or salts thereof, may be formulated as pharmaceutical compositions prepared for storage or administration, which typically comprise a therapeutically effective amount of a compound of the present invention, or a salt thereof, in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences (Mack Publishing Co. (A. Gennaro, ed., 1985 ).
  • pharmaceutically acceptable carrier includes any and all physiologically acceptable, i.e. compatible, solvents, dispersion media, coatings, antimicrobial agents, isotonic, and absorption delaying agents, and the like.
  • compositions of the invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. In such form, the composition is divided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparations, for example, packeted tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms. It may be provided in single dose injectable form, for example in the form of a pen.
  • Compositions may be formulated for any suitable route and means of administration. Subcutaneous or transdermal modes of administration may be particularly suitable for therapeutic proteins described herein.
  • a pharmaceutical composition of the present invention also optionally includes a pharmaceutically acceptable antioxidant.
  • exemplary pharmaceutically acceptable antioxidants are water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (
  • the present invention provides pharmaceutical compositions comprising one or a combination of different polypeptides and/or cell-targeted molecules of the invention, or an ester, salt or amide of any of the foregoing, and at least one pharmaceutically acceptable carrier.
  • compositions are typically sterile and stable under the conditions of manufacture and storage.
  • the composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier may be a solvent or dispersion medium containing, for example, water, alcohol such as ethanol, polyol (e.g. glycerol, propylene glycol, and liquid polyethylene glycol), or any suitable mixtures.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by use of surfactants according to formulation chemistry well known in the art.
  • isotonic agents e.g.
  • compositions sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride may be desirable in the composition.
  • Prolonged absorption of injectable compositions may be brought about by including in the composition an agent that delays absorption for example, monostearate salts and gelatin.
  • Solutions or suspensions used for intradermal or subcutaneous application typically include one or more of: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonicity adjusting agents such as, e.g., sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide, or buffers with citrate, phosphate, acetate and the like.
  • acids or bases such as hydrochloric acid or sodium hydroxide, or buffers with citrate, phosphate, acetate and the like.
  • Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Sterile injectable solutions may be prepared by incorporating a polypeptide or cell-targeted molecule of the invention in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, followed by sterilization microfiltration.
  • Dispersions may be prepared by incorporating the active compound into a sterile vehicle that contains a dispersion medium and other ingredients, such as those described above.
  • the methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient in addition to any additional desired ingredient from a sterile-filtered solution thereof.
  • the binding agent When a therapeutically effective amount of a polypeptide or cell-targeted molecule of the invention is designed to be administered by, e.g. intravenous, cutaneous or subcutaneous injection, the binding agent will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. Methods for preparing parenterally acceptable protein solutions, taking into consideration appropriate pH, isotonicity, stability, and the like, are within the skill in the art.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection will contain, in addition to binding agents, an isotonic vehicle such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicle as known in the art.
  • a pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives well known to those of skill in the art.
  • a polypeptide or cell-targeted molecule of the invention may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art (see e.g. Sustained and Controlled Release Drug Delivery Systems (Robinson J, ed., Marcel Dekker, Inc., NY, U.S., 1978 )).
  • the pharmaceutical composition of the present invention may be formulated to ensure a desired distribution in vivo.
  • the blood-brain barrier excludes many large and/or hydrophilic compounds.
  • they can be formulated, for example, in liposomes which may comprise one or more moieties that are selectively transported into specific cells or organs, thus enhancing targeted drug delivery.
  • exemplary targeting moieties include folate or biotin; mannosides; antibodies; surfactant protein A receptor; p120 catenin and the like.
  • compositions include parenteral formulations designed to be used as implants or particulate systems.
  • implants are depot formulations composed of polymeric or hydrophobic components such as emulsions, ion exchange resins, and soluble salt solutions.
  • particulate systems are microspheres, microparticles, nanocapsules, nanospheres, and nanoparticles (see e.g. Honda M et al., Int J Nanomedicine 8: 495-503 (2013 ); Sharma A et al., Biomed Res Int 2013: 960821 (2013 ); Ramishetti S, Huang L, Ther Deliv 3: 1429-45 (2012 )).
  • Controlled release formulations may be prepared using polymers sensitive to ions, such as, e.g. liposomes, polaxamer 407, and hydroxyapatite.
  • polynucleotide is equivalent to the term "nucleic acid,” each of which includes one or more of: polymers of deoxyribonucleic acids (DNAs), polymers of ribonucleic acids (RNAs), analogs of these DNAs or RNAs generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • the polynucleotide of the present invention may be single-, double-, or triple-stranded.
  • Such polynucleotides are specifically disclosed to include all polynucleotides capable of encoding an exemplary protein, for example, taking into account the wobble known to be tolerated in the third position of RNA codons, yet encoding for the same amino acid as a different RNA codon (see Stothard P, Biotechniques 28: 1102-4 (2000 )).
  • the invention provides polynucleotides which encode a CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide and/or cell-targeted protein of the invention, or a fragment or derivative thereof.
  • the polynucleotides may include, e.g., nucleic acid sequence encoding a polypeptide at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, identical to a polypeptide comprising one of the amino acid sequences of the protein.
  • the invention also includes polynucleotides comprising nucleotide sequences that hybridize under stringent conditions to a polynucleotide which encodes CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide and/or cell-targeted protein of the invention, or a fragment or derivative thereof, or the antisense or complement of any such sequence.
  • Derivatives or analogs of the molecules (e.g., CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides and/or cell-targeted proteins comprising the same) of the present invention include, inter alia, polynucleotide (or polypeptide) molecules having regions that are substantially homologous to the polynucleotides, CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides, or cell-targeted proteins of the present invention, e.g.
  • polynucleotides capable of hybridizing to the complement of a sequence encoding the cell-targeted proteins of the invention under stringent conditions (see e.g. Ausubel F et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York, NY, U.S., 1993 )), and below. Stringent conditions are known to those skilled in the art and may be found, e.g., in Current Protocols in Molecular Biology (John Wiley & Sons, NY, U.S., Ch. Sec. 6.3.1-6.3.6 (1989 )).
  • Such expression vectors will include the polynucleotides necessary to support production of contemplated CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides and/or cell-targeted proteins of the invention within any host cell of choice or cell-free expression systems (e.g. pTxb1 and pIVEX2.3 described in the Examples below).
  • the specific polynucleotides comprising expression vectors for use with specific types of host cells or cell-free expression systems are well known to one of ordinary skill in the art, can be determined using routine experimentation, or may be purchased.
  • expression vector refers to a polynucleotide, linear or circular, comprising one or more expression units.
  • expression unit denotes a polynucleotide segment encoding a polypeptide of interest and capable of providing expression of the nucleic acid segment in a host cell.
  • An expression unit typically comprises a transcription promoter, an open reading frame encoding the polypeptide of interest, and a transcription terminator, all in operable configuration.
  • An expression vector contains one or more expression units.
  • an expression vector encoding a CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide and/or protein comprising a single polypeptide chain includes at least an expression unit for the single polypeptide chain, whereas a protein comprising, e.g. two or more polypeptide chains (e.g .
  • one chain comprising a V L domain and a second chain comprising a V H domain linked to a toxin effector region includes at least two expression units, one for each of the two polypeptide chains of the protein.
  • an expression unit for each polypeptide chain may also be separately contained on different expression vectors (e.g. expression may be achieved with a single host cell into which expression vectors for each polypeptide chain has been introduced).
  • Expression vectors capable of directing transient or stable expression of polypeptides and proteins are well known in the art.
  • the expression vectors generally include, but are not limited to, one or more of the following: a heterologous signal sequence or peptide, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, each of which is well known in the art.
  • Optional regulatory control sequences, integration sequences, and useful markers that can be employed are known in the art.
  • host cell refers to a cell which can support the replication or expression of the expression vector.
  • Host cells may be prokaryotic cells, such as E. coli or eukaryotic cells (e.g. yeast, insect, amphibian, bird, or mammalian cells). Creation and isolation of host cell lines comprising a polynucleotide of the invention or capable of producing a polypeptide and/or cell-targeted protein of the invention can be accomplished using standard techniques known in the art.
  • the invention relates to a device comprising one or more compositions of matter of the invention, such as a pharmaceutical composition, for delivery to a subject in need thereof.
  • a delivery device comprising one or more compounds of the invention can be used to administer to a patient a composition of matter of the invention by various delivery methods, including: intravenous, subcutaneous, intramuscular or intraperitoneal injection; oral administration; transdermal administration; pulmonary or transmucosal administration; administration by implant, osmotic pump, cartridge or micro pump; or by other means recognized by a person of skill in the art.
  • kits comprising at least one composition of matter of the invention, and optionally, packaging and instructions for use.
  • Kits may be useful for drug administration and/or diagnostic information gathering.
  • a kit of the invention may optionally comprise at least one additional reagent (e.g. , standards, markers and the like).
  • Kits typically include a label indicating the intended use of the contents of the kit.
  • the kit may further comprise reagents and other tools for detecting a cell type (e.g. tumor cell) in a sample or in a subject, or for diagnosing whether a patient belongs to a group that responds to a therapeutic strategy which makes use of a compound, composition or related method of the invention as described herein.
  • a polypeptide already capable of intracellularly routing to a cytosol, ER, or lysosome of a cell from an endosomal compartment of the cell is created into a T-cell hyper-immunized polypeptide of the present invention; the method comprising the step of adding a heterologous T-cell epitope to the polypeptide.
  • a polypeptide capable of delivering a T-cell epitope for presentation by a MHC class I molecule comprising the step of adding a heterologous T-cell epitope to a polypeptide capable of intracellular delivery of the T-cell epitope from an endosomal compartment of a cell to a proteasome of the cell.
  • the heterologous T-cell epitope is embedded or inserted within a polypeptide capable of intracellularly routing to a cytosol, ER, or lysosome of a cell from an endosomal compartment of the cell.
  • a T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide is created; the method comprising the step of inserting or embedding a heterologous T-cell epitope into an endogenous B-cell epitope region of a polypeptide already capable of intracellularly routing to a cytosol, ER, or lysosome of a cell from an endosomal compartment of the cell.
  • a CD8+ T-cell hyper-immunized and B-cell/CD4+ T-cell de-immunized polypeptide of the present invention is created; the method comprising the step of embedding or inserting a heterologous T-cell epitope into an endogenous B-cell epitope region of a polypeptide already capable of intracellularly routing to a cytosol, ER, or lysosome of a cell from an endosomal compartment of the cell.
  • a polypeptide already capable of intracellularly routing to a cytosol, ER, or lysosome of a cell from an endosomal compartment of the cell is created into a T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide of the present invention; the method comprising the step of embedding or inserting a heterologous T-cell epitope into an endogenous B-cell epitope region of the polypeptide.
  • a polypeptide already capable of intracellularly routing to a cytosol, ER, or lysosome of a cell from an endosomal compartment of the cell is created into a CD8+ T-cell hyper-immunized polypeptide of the present invention; the method comprising the step of embedding or inserting a heterologous T-cell epitope into an endogenous B-cell epitope region of the polypeptide.
  • a de-immunized polypeptide capable of delivering a T-cell epitope for presentation by a MHC class I molecule is created; the method comprising the step of embedding or inserting a heterologous T-cell epitope into an endogenous B-cell epitope region of a polypeptide capable of intracellular delivery of the T-cell epitope from an endosomal compartment of a cell to a proteasome of the cell.
  • a de-immunized polypeptide is created which has reduced B-cell immunogenicity when administered to a chordate.
  • is a method for reducing B-cell immunogenicity in a polypeptide comprising the step of disrupting a B-cell epitope region within a polypeptide with one or more amino acid residue(s) comprised by a heterologous T-cell epitope added to the polypeptide.
  • the disrupting step further comprises creating one or more amino acid substitutions in the B-cell epitope region.
  • the disrupting step further comprises creating one or more amino acid insertions in the B-cell epitope region.
  • Certain embodiments of the methods of the present invention are methods for reducing B-cell immunogenicity in a polypeptide while simultaneously increasing CD8+ T-cell immunogenicity after administration to a chordate, the methods comprising the step of disrupting a B-cell epitope region within a polypeptide with one or more amino acid residue(s) comprised by a heterologous CD8+ T-cell epitope added to the polypeptide.
  • the disrupting step further comprises creating one or more amino acid substitutions in the B-cell epitope region.
  • the disrupting step further comprises creating one or more amino acid insertions in the B-cell epitope region.
  • Certain embodiments of the methods of the present invention are methods for reducing B-cell immunogenicity in a polypeptide while simultaneously increasing CD8+ T-cell immunogenicity after administration to a chordate, the methods comprising the steps of: 1) identifying a B-cell epitope in a polypeptide; and 2) disrupting the identified B-cell epitope with one or more amino acid residue(s) comprised by a heterologous CD8+ T-cell epitope added to the polypeptide.
  • the disrupting step further comprises the creation of one or more amino acid substitutions in the B-cell epitope region.
  • the disrupting step further comprises creating one or more amino acid insertions in the B-cell epitope region.
  • Certain embodiments of the methods of the present invention are methods for reducing B-cell immunogenicity in a polypeptide while simultaneously increasing CD8+ T-cell immunogenicity after administration to a chordate, the methods comprising the steps of: 1) identifying a B-cell epitope in a polypeptide; and 2) disrupting the identified B-cell epitope with one or more amino acid residue(s) comprised by a heterologous CD8+ T-cell epitope added to the polypeptide.
  • the disrupting step further comprises the creation of one or more amino acid substitutions in the B-cell epitope region.
  • the disrupting step further comprises creating one or more amino acid insertions in the B-cell epitope region.
  • a CD4+ T-cell de-immunized polypeptide is created which has reduced CD4+ T-cell immunogenicity when administered to a chordate.
  • a method for reducing CD4+ T-cell immunogenicity in a polypeptide comprising the step of disrupting a CD4+ T-cell epitope region within a polypeptide with one or more amino acid residue(s) comprised by a heterologous CD8+ T-cell epitope added to the polypeptide.
  • the disrupting step further comprises creating one or more amino acid substitutions in the B-cell epitope region.
  • the disrupting step further comprises creating one or more amino acid insertions in the CD4+ T-cell epitope region.
  • Certain embodiments of the methods of the present invention are methods for reducing CD4+ T-cell immunogenicity in a polypeptide while simultaneously increasing CD8+ T-cell immunogenicity after administration to a chordate, the methods comprising the step of disrupting a CD4+ T-cell epitope region within a polypeptide with one or more amino acid residue(s) comprised by a heterologous CD8+ T-cell epitope added to the polypeptide.
  • the disrupting step further comprises creating one or more amino acid substitutions in the CD4+ T-cell epitope region.
  • the disrupting step further comprises creating one or more amino acid insertions in the CD4+ T-cell epitope region.
  • Certain embodiments of the methods of the present invention are methods for reducing CD4+ T-cell immunogenicity in a polypeptide while simultaneously increasing CD8+ T-cell immunogenicity after administration to a chordate, the methods comprising the steps of: 1) identifying a CD4+ T-cell epitope in a polypeptide; and 2) disrupting the identified CD4+ T-cell epitope with one or more amino acid residue(s) comprised by a heterologous CD8+ T-cell epitope added to the polypeptide.
  • the disrupting step further comprises the creation of one or more amino acid substitutions in the CD4+ T-cell epitope region.
  • the disrupting step further comprises creating one or more amino acid insertions in the CD4+ T-cell epitope region.
  • Certain embodiments of the methods of the present invention are methods for reducing CD4+ T-cell immunogenicity in a polypeptide while simultaneously increasing CD8+ T-cell immunogenicity after administration to a chordate, the methods comprising the steps of: 1) identifying a CD4+ T-cell epitope in a polypeptide; and 2) disrupting the identified CD4+ T-cell epitope with one or more amino acid residue(s) comprised by a heterologous CD8+ T-cell epitope added to the polypeptide.
  • the disrupting step further comprises the creation of one or more amino acid substitutions in the CD4+ T-cell epitope region.
  • the disrupting step further comprises creating one or more amino acid insertions in the CD4+ T-cell epitope region.
  • cytotoxic molecules such as e.g . potential therapeutics comprising cytotoxic toxin region polypeptides
  • cytotoxic toxin region polypeptides may be engineered to be more cytotoxic and/or to have redundant, backup cytotoxicities operating via completely different mechanisms.
  • These multiple cytotoxic mechanisms may complement each other (such as by providing both two mechanisms of cell killing, direct and indirect, as well as mechanisms of immuno-stimulation to the local area), redundantly backup each other (such as by providing direct cell killing in the absence of the other), and/or protect against developed resistance (by limiting resistance to the less probable situation of the malignant or infected cell blocking two different mechanisms simultaneously).
  • parental cytotoxic molecules which rely on toxin effector and/or enzymatic regions for cytotoxicity may be engineered by mutating the parental molecule to be enzymatically inactive but to be cytotoxic via T-cell epitope delivery to the MHC class I system of a target cell and subsequent presentation to the surface of the target cell. This approach removes one cytotoxic mechanism while adding another and adds the capability of immuno-stimulation to the local area of the target cell by T-cell epitope presentation.
  • parental cytotoxic molecules which rely on enzymatic regions for cytotoxicity may be engineered to be cytotoxic only via T-cell epitope delivery to the MHC class I system by embedding a T-cell epitope in the enzymatic domain of the parental molecule such that the enzymatic activity is reduced or eliminated.
  • This allows for the one-step modification of enzymatically-cytotoxic molecules, which have the ability once in an endosomal compartment to route to the cytosol and/or ER, into enzymatically inactive, cytotoxic molecules which rely on T-cell epitope delivery to the MHC class I system of a target cell and subsequent presentation on the surface of the target cell for cytotoxicity.
  • the present invention provides methods of using polypeptides and proteins with characterized by polypeptide sequences and pharmaceutical compositions thereof.
  • polypeptide sequences in SEQ ID NOs: 1-60 may be specifically utilized as a component of the cell-targeted molecules used in the following methods.
  • the present invention provides methods of killing a cell comprising the step of contacting the cell, either in vitro or in vivo, with a polypeptide, protein, or pharmaceutical composition of the present invention.
  • the polypeptides, proteins, and pharmaceutical compositions of the present invention can be used to kill a specific cell type upon contacting a cell or cells with one of the claimed compositions of matter.
  • a cytotoxic polypeptide, protein, or pharmaceutical composition of the present invention can be used to kill specific cell types in a mixture of different cell types, such as mixtures comprising cancer cells, infected cells, and/or hematological cells.
  • a cytotoxic polypeptide, protein, or pharmaceutical composition of the present invention can be used to kill cancer cells in a mixture of different cell types.
  • a cytotoxic polypeptide, protein, or pharmaceutical composition of the present invention can be used to kill specific cell types in a mixture of different cell types, such as pre-transplantation tissues.
  • a polypeptide, protein, or pharmaceutical composition of the present invention can be used to kill specific cell types in a mixture of cell types, such as pre-administration tissue material for therapeutic purposes.
  • a polypeptide, protein, or pharmaceutical composition of the present invention can be used to selectively kill cells infected by viruses or microorganisms, or otherwise selectively kill cells expressing a particular extracellular target biomolecule, such as a cell surface biomolecule.
  • the polypeptides, proteins, and pharmaceutical compositions of the present invention have varied applications, including, e.g ., uses in depleting unwanted cell types from tissues either in vitro or in vivo, uses in modulating immune responses to treat graft versus host, uses as antiviral agents, uses as anti-parasitic agents, and uses in purging transplantation tissues of unwanted cell types.
  • a cytotoxic polypeptide, protein, or pharmaceutical composition of the present invention can show potent cell-kill activity when administered to a population of cells, in vitro or in vivo in a subject such as in a patient in need of treatment.
  • this potent cell-kill activity can be restricted to specifically and selectively kill certain cell types within an organism, such as certain cancer cells, neoplastic cells, malignant cells, non-malignant tumor cells, or infected cells.
  • the present invention provides a method of killing a cell in a patient in need thereof, the method comprising the step of administering to the patient at least one cytotoxic polypeptide or protein of the present invention, or a pharmaceutical composition thereof.
  • cytotoxic polypeptide, protein, or pharmaceutical compositions thereof can be used to kill a cancer cell in a patient by targeting an extracellular biomolecule found physically coupled with a cancer or tumor cell.
  • cancer cell or “cancerous cell” refers to various neoplastic cells which grow and divide in an abnormally accelerated fashion and will be clear to the skilled person.
  • tumor cell includes both malignant and non-malignant cells.
  • cancers and/or tumors can be defined as diseases, disorders, or conditions that are amenable to treatment and/or prevention.
  • cytotoxic polypeptide or cell-targeted molecule of the present invention can be used to kill an immune cell (whether healthy or malignant) in a patient by targeting an extracellular biomolecule found physically coupled with an immune cell.
  • the cell-targeted molecule of the present invention or pharmaceutical composition thereof for the purposes of purging patient cell populations (e.g. bone marrow) of malignant, neoplastic, or otherwise unwanted T-cells and/or B-cells and then reinfusing the T-cell and/or B-cells depleted material into the patient (see e.g. van Heeckeren W et al., Br J Haematol 132: 42-55 (2006 ); (see e.g. Alpdogan O, van den Brink M, Semin Oncol 39: 629-42 (2012 )).
  • the cell-targeted molecule of the present invention can be used in a method for prophylaxis of organ and/or tissue transplant rejection wherein the donor organ or tissue is perfused prior to transplant with a cytotoxic, cell-targeted molecule of the invention or a pharmaceutical composition thereof in order to purge the organ of donor T-cells and/or B-cells (see e.g. Alpdogan O, van den Brink M, Semin Oncol 39: 629-42 (2012 )).
  • cytotoxic polypeptide or cell-targeted molecule of the invention can be used to kill an infected cell in a patient by targeting an extracellular biomolecule found physically coupled with an infected cell.
  • the non-self, T-cell epitope-peptide is selected from the group consisting of: peptides not already presented by the target cells of the cell-targeted molecule, peptides not present within any protein expressed by the target cell, peptides not present within the proteome of the target cell, peptides not present in the extracellular microenvironment of the site to be seeded, and peptides not present in the tumor mass or infect tissue site to be targeted.
  • This "seeding" method functions to label one or more target cells within a chordate with one or more MHC class I presented T-cell epitopes for recognition by effector T-cells and activation of downstream immune responses.
  • the target cells which display the delivered T-cell epitope are harnessed to induce recognition of the presenting target cell by host T-cells and induction of further immune responses including target cell killing by CTLs.
  • the therapeutically effective amount of a compound of the present invention will depend on the route of administration, the type of mammal being treated, and the physical characteristics of the specific patient under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical arts. This amount and the method of administration can be tailored to achieve optimal efficacy, and may depend on such factors as weight, diet, concurrent medication and other factors, well known to those skilled in the medical arts.
  • the dosage sizes and dosing regimen most appropriate for human use may be guided by the results obtained by the present invention, and may be confirmed in properly designed clinical trials.
  • An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Such considerations are known to the skilled person.
  • An acceptable route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal (e.g. topical administration of a cream, gel or ointment, or by means of a transdermal patch).
  • Parental administration is typically associated with injection at or in communication with the intended site of action, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal administration.
  • the dosage range will generally be from about 0.0001 to 100 milligrams per kilogram (mg/kg), and more, usually 0.01 to 5 mg/kg, of the host body weight.
  • Exemplary dosages may be 0.25 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.
  • An exemplary treatment regime is a once or twice daily administration, or a once or twice weekly administration, once every two weeks, once every three weeks, once every four weeks, once a month, once every two or three months or once every three to 6 months. Dosages may be selected and readjusted by the skilled health care professional as required to maximize therapeutic benefit for a particular patient.
  • compositions of the present invention will typically be administered to the same patient on multiple occasions. Intervals between single dosages can be, for example, 2-5 days, weekly, monthly, every two or three months, every six months, or yearly. Intervals between administrations can also be irregular, based on regulating blood levels or other markers in the subject or patient. Dosage regimens for a compound of the invention include intravenous administration of 1 mg/kg body weight or 3 mg/kg body weight with the compound administered every two to four weeks for six dosages, then every three months at 3 mg/kg body weight or 1 mg/kg body weight.
  • a pharmaceutical composition of the present invention may be administered via one or more routes of administration, using one or more of a variety of methods known in the art. As will be appreciated by the skilled worker, the route and/or mode of administration will vary depending upon the desired results. Routes of administration for polypeptides, proteins, and pharmaceutical compositions of the invention include, e.g . intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, for example by injection or infusion.
  • a polypeptide, protein, or pharmaceutical composition of the invention may be administered by a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.
  • a non-parenteral route such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.
  • Therapeutic polypeptides, proteins, or pharmaceutical compositions of the present invention may be administered with one or more of a variety of medical devices known in the art.
  • a pharmaceutical composition of the invention may be administered with a needleless hypodermic injection device.
  • implants and modules useful in the present invention are in the art, including e.g ., implantable micro-infusion pumps for controlled rate delivery; devices for administering through the skin; infusion pumps for delivery at a precise infusion rate; variable flow implantable infusion devices for continuous drug delivery; and osmotic drug delivery systems. These and other such implants, delivery systems, and modules are known to those skilled in the art.
  • a polypeptide, protein, or pharmaceutical composition of the present invention may be administered alone or in combination with one or more other therapeutic or diagnostic agents.
  • a combination therapy may include a cytotoxic, cell-targeted molecule of the invention or pharmaceutical composition thereof combined with at least one other therapeutic agent selected based on the particular patient, disease or condition to be treated.
  • agents include, inter alia, a cytotoxic, anti-cancer or chemotherapeutic agent, an anti-inflammatory or anti-proliferative agent, an antimicrobial or antiviral agent, growth factors, cytokines, an analgesic, a therapeutically active small molecule or polypeptide, a single chain antibody, a classical antibody or fragment thereof, or a nucleic acid molecule which modulates one or more signaling pathways, and similar modulating therapeutics which may complement or otherwise be beneficial in a therapeutic or prophylactic treatment regimen.
  • Treatment of a patient with a polypeptide, protein, or pharmaceutical composition of the present invention preferably leads to cell death of targeted cells and/or the inhibition of growth of targeted cells.
  • cytotoxic, cell-targeted molecules of the present invention, and pharmaceutical compositions comprising them will be useful in methods for treating a variety of pathological disorders in which killing or depleting target cells may be beneficial, such as, inter alia, cancer, tumors, other growth abnormalities, immune disorders, and infected cells.
  • the present invention provides methods for suppressing cell proliferation, and treating cell disorders, including neoplasia, overactive B-cells, and overactive T-cells.
  • polypeptides, proteins, and pharmaceutical compositions of the present invention can be used to treat or prevent cancers, tumors (malignant and non-malignant), growth abnormalities, immune disorders, and microbial infections.
  • the above ex vivo method can be combined with the above in vivo method to provide methods of treating or preventing rejection in bone marrow transplant recipients, and for achieving immunological tolerance.
  • the present invention provides methods for treating malignancies or neoplasms and other blood cell associated cancers in a mammalian subject, such as a human, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a cytotoxic protein or pharmaceutical composition of the invention.
  • the cytotoxic polypeptides, proteins, and pharmaceutical compositions of the present invention have varied applications, including, e.g ., uses in removing unwanted T-cells, uses in modulating immune responses to treat graft versus host, uses as antiviral agents, uses as antimicrobial agents, and uses in purging transplantation tissues of unwanted cell types.
  • the cytotoxic polypeptides, proteins, and pharmaceutical compositions of the present invention are commonly anti-neoplastic agents - meaning they are capable of treating and/or preventing the development, maturation, or spread of neoplastic or malignant cells by inhibiting the growth and/or causing the death of cancer or tumor cells.
  • a polypeptide, protein, or pharmaceutical composition of the present invention is used to treat a B-cell-, plasma cell- or antibody- mediated disease or disorder, such as for example leukemia, lymphoma, myeloma, Human Immunodeficiency Virus-related diseases, amyloidosis, hemolytic uremic syndrome, polyarteritis, septic shock, Crohn's Disease, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, ulcerative colitis, psoriasis, asthma, Sjorgren's syndrome, graft-versus-host disease, graft rejection, diabetes, vasculitis, scleroderma, and systemic lupus erythematosus.
  • a B-cell-, plasma cell- or antibody- mediated disease or disorder such as for example leukemia, lymphoma, myeloma, Human Immunodeficiency Virus-related diseases, amyloidosis,
  • certain embodiments of the polypeptides, proteins, and pharmaceutical compositions of the present invention are antimicrobial agents - meaning they are capable of treating and/or preventing the acquisition, development, or consequences of microbiological pathogenic infections, such as caused by viruses, bacteria, fungi, prions, or protozoans.
  • T-cells or B-cells by administering the cytotoxic protein or the invention, or a pharmaceutical composition thereof, to a patient for the purpose of killing T-cells or B-cells in the patient.
  • This usage is compatible with preparing or conditioning a patient for bone marrow transplantation, stem cell transplantation, tissue transplantation, or organ transplantation, regardless of the source of the transplanted material, e.g . human or non-human sources.
  • cytotoxic polypeptides, proteins, and pharmaceutical compositions of the present invention can be utilized in a method of treating cancer comprising administering to a patient, in need thereof, a therapeutically effective amount of a cytotoxic polypeptide, protein, or pharmaceutical composition of the present invention.
  • the cancer being treated is selected from the group consisting of: bone cancer (such as multiple myeloma or Ewing's sarcoma), breast cancer, central/peripheral nervous system cancer (such as brain cancer, neurofibromatosis, or glioblastoma), gastrointestinal cancer (such as stomach cancer or colorectal cancer), germ cell cancer (such as ovarian cancers and testicular cancers, glandular cancer (such as pancreatic cancer, parathyroid cancer, pheochromocytoma, salivary gland cancer, or thyroid cancer), head-neck cancer (such as nasopharyngeal cancer, oral cancer, or pharyngeal cancer), hematological cancers (such as leukemia, lymphoma, or myeloma), kidney-urinary tract cancer (such as renal cancer and bladder cancer), liver cancer, lung/pleura cancer (such as mesothelioma, small cell lung carcinoma, or non-small cell lung carcinoma),
  • bone cancer such as multiple myelo
  • the polypeptides, proteins, and pharmaceutical compositions of the present invention can be utilized in a method of treating an immune disorder comprising administering to a patient, in need thereof, a therapeutically effective amount of the cytotoxic protein or a pharmaceutical composition of the present invention.
  • the immune disorder is related to an inflammation associated with a disease selected from the group consisting of: amyloidosis, ankylosing spondylitis, asthma, Crohn's disease, diabetes, graft rejection, graft-vs.-host disease, Hashimoto's thyroiditis, hemolytic uremic syndrome, HIV-related diseases, lupus erythematosus, multiple sclerosis, polyarteritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, septic shock, Sjorgren's syndrome, ulcerative colitis, and vasculitis.
  • a disease selected from the group consisting of: amyloidosis, ankylosing s
  • the polypeptide or cell-targeted molecule of the present invention is using the polypeptide or cell-targeted molecule of the present invention as a component of a pharmaceutical composition or medicament for the treatment or prevention of a cancer, tumor, other growth abnormality, immune disorder, and/or microbial infection.
  • immune disorders presenting on the skin of a patient may be treated with such a medicament in efforts to reduce inflammation.
  • skin tumors may be treated with such a medicament in efforts to reduce tumor size or eliminate the tumor completely.
  • certain cytotoxic, cell-targeted molecules of the invention may be used to kill a neuron(s) which expresses the extracellular target at a site of cytotoxic protein injection distant from the cell body ( see Llewellyn-Smith I et al., J Neurosci Methods 103: 83-90 (2000 )).
  • These neuronal cell type specific targeting cytotoxic polypeptides and proteins have uses in neuroscience research, such as for elucidating mechanisms of sensations ( see e.g. Mishra S, Hoon M, Science 340: 968-71 (2013 ), and creating model systems of neurodegenerative diseases, such as Parkinson's and Alzheimer's (see e.g. Hamlin A et al., PLoS One e53472 (2013 )).
  • a method of using a polypeptide, protein, pharmaceutical composition, and/or diagnostic composition of the present invention to label or detect the interiors of neoplastic cells and/or immune cell types. Based on the ability of certain polypeptides, proteins, and pharmaceutical compositions of the invention to enter specific cell types and route within cells via retrograde intracellular transport, the interior compartments of specific cell types are labeled for detection. This can be performed on cells in situ within a patient or on cells and tissues removed from an organism, e.g . biopsy material.
  • the present invention is a method of using a polypeptide, protein, pharmaceutical composition, and/or diagnostic composition of the present invention to detect the presence of a cell type for the purpose of information gathering regarding diseases, conditions and/or disorders.
  • the method comprises contacting a cell with a diagnostically sufficient amount of a cytotoxic molecule to detect the cytotoxic molecule by an assay or diagnostic technique.
  • diagnostically sufficient amount refers to an amount that provides adequate detection and accurate measurement for information gathering purposes by the particular assay or diagnostic technique utilized.
  • the diagnostically sufficient amount for whole organism in vivo diagnostic use will be a non-cumulative dose of between 0.1 mg to 100 mg of the detection promoting agent linked cell-targeted molecule of the invention per kg of subject per subject.
  • the amount of polypeptide or cell-targeted molecule of the invention used in these information gathering methods will be as low as possible provided that it is still a diagnostically sufficient amount.
  • the amount of polypeptide, protein, or pharmaceutical composition of the invention administered to a subject will be as low as feasibly possible.
  • various detection promoting agents may be utilized for non-invasive in vivo tumor imaging by techniques such as magnetic resonance imaging (MRI), optical methods (such as direct, fluorescent, and bioluminescent imaging), positron emission tomography (PET), single-photon emission computed tomography (SPECT), ultrasound, x-ray computed tomography, and combinations of the aforementioned ( see, Kaur S et al., Cancer Lett 315: 97-111 (2012 ), for review).
  • MRI magnetic resonance imaging
  • optical methods such as direct, fluorescent, and bioluminescent imaging
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • ultrasound x-ray computed tomography
  • a polypeptide, protein, or pharmaceutical composition of the present invention is a method of using a polypeptide, protein, or pharmaceutical composition of the present invention as a diagnostic composition to label or detect the interiors of cancer, tumor, and/or immune cell types (see e.g., Koyama Y et al., Clin Cancer Res 13: 2936-45 (2007 ); Ogawa M et al., Cancer Res 69: 1268-72 (2009 ); Yang L et al., Small 5: 235-43 (2009 )).
  • the interior compartments of specific cell types are labeled for detection. This can be performed on cells in situ within a patient or on cells and tissues removed from an organism, e.g . biopsy material.
  • Diagnostic compositions of the present invention may be used to characterize a disease, disorder, or condition as potentially treatable by a related pharmaceutical composition of the present invention. Certain compositions of matter of the present invention may be used to determine whether a patient belongs to a group that responds to a therapeutic strategy which makes use of a compound, composition or related method of the present invention as described herein or is well suited for using a delivery device of the invention.
  • Diagnostic compositions of the present invention may be used after a disease, e.g. a cancer, is detected in order to better characterize it, such as to monitor distant metastases, heterogeneity, and stage of cancer progression.
  • a disease e.g. a cancer
  • the phenotypic assessment of disease disorder or infection can help prognostic and prediction during therapeutic decision making.
  • certain methods of the invention may be used to determine if local or systemic problem.
  • Diagnostic compositions of the present invention may be used to assess responses to therapeutic(s) regardless of the type of therapeutic, e.g . small molecule drug, biological drug, or cell-based therapy.
  • certain embodiments of the diagnostics of the invention may be used to measure changes in tumor size, changes in antigen positive cell populations including number and distribution, or monitoring a different marker than the antigen targeted by a therapy already being administered to a patient (see Smith-Jones P et al., Nat. Biotechnol 22: 701-6 (2004 ); Evans M et al., Proc. Natl. Acad. Sci. U.S.A. 108: 9578-82 (2011 )).
  • Certain embodiments of the method used to detect the presence of a cell type may be used to gather information regarding diseases, disorders, and conditions, such as, for example bone cancer (such as multiple myeloma or Ewing's sarcoma), breast cancer, central/peripheral nervous system cancer (such as brain cancer, neurofibromatosis, or glioblastoma), gastrointestinal cancer (such as stomach cancer or colorectal cancer), germ cell cancer (such as ovarian cancers and testicular cancers, glandular cancer (such as pancreatic cancer, parathyroid cancer, pheochromocytoma, salivary gland cancer, or thyroid cancer), head-neck cancer (such as nasopharyngeal cancer, oral cancer, or pharyngeal cancer), hematological cancers (such as leukemia, lymphoma, or myeloma), kidney-urinary tract cancer (such as renal cancer and bladder cancer), liver cancer, lung/pleura cancer (such as mesothelioma, small cell
  • the polypeptides and cell-targeted molecules of the present invention are used for both diagnosis and treatment, or for diagnosis alone.
  • the present invention is further illustrated by the following non-limiting examples of 1) CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides, 2) CD8+ T-cell epitope presenting toxin-derived polypeptides, and 3) selectively cytotoxic, cell-targeted proteins comprising the aforementioned polypeptides and capable of specifically targeting certain cell types.
  • Polypeptide effectors derived from both a Shiga toxin and a diphtheria toxin were analyzed for B-cell epitopes.
  • Ten different polynucleotides were generated which each encoded for a different exemplary cytotoxic, cell-targeted protein of the invention comprising a different exemplary Shiga toxin effector polypeptide component of the invention. These exemplary polynucleotides were used to produce ten exemplary cytotoxic, cell-targeted proteins of the invention using standard techniques known in the art. In certain experiments, the full-length coding sequence of the cytotoxic protein of this example began or ended with a polynucleotide encoding a Strep-tag® II to facilitate detection and purification. Proteins were purified using methods known to the skilled worker.
  • Shiga toxin effector polypeptides represented by SEQ ID NOs:11-17 amd 19-21 all comprise a disruption of a predicted endogenous CD4+ T-cell epitope(s).
  • T-cell epitope embedded, diphtheria toxin effector polypeptide variants SEQ ID NOs: 46, 47, and 48
  • parental diphtheria toxin effector polypeptide comprising only wild-type toxin amino acid sequences
  • SEQ ID NO:45 Three T-cell epitope embedded, diphtheria toxin effector polypeptide variants (SEQ ID NOs: 46, 47, and 48), and the parental diphtheria toxin effector polypeptide comprising only wild-type toxin amino acid sequences (SEQ ID NO:45), were each designed with an amino-terminal methionine and a carboxy-terminal polyhistidine-tag (6xHis tag) to facilitate expression and purification.
  • T-cell epitope embedded, diphtheria toxin effector polypeptide variants of the invention and the parental diphtheria toxin effector polypeptide comprising only wild-type toxin amino acid sequences were produced by a bacterial system known in the art and purified under conditions known in the art, such as, e.g ., nickel-nitrilotriacetic acid (Ni-NTA) resin chromatography.
  • Ni-NTA nickel-nitrilotriacetic acid
  • T-cell epitope peptide sequences from Table 1 are embedded in those regions identified to lack B-cell epitopes by replacing the native amino acids by substitutions to create three different exemplary Shiga toxin effector polypeptides of the invention as shown in Table 8.
  • Table 8 shows the identified regions in the mature, native SLT-1A polypeptide sequence and the replacement T-cell epitope sequences constructed into the Shiga toxin effector polypeptides (see SEQ ID NOs: 22-39). Table 8.
  • Shiga toxin effector polypeptides represented by SEQ ID NOs: 22-39 all comprise a disruption of a predicted endogenous CD4+ T-cell epitope(s) except for the polypeptides with heterologous T-cell epitopes embedded at position 221-229, which are represented by SEQ ID NOs: 26, 32, and 38.
  • Table 9 shows the position of the embedded T-cell epitopes in the mature, native SLT-1A polypeptide sequence, the replacement T-cell epitope sequences which are embedded, the replaced sequences in the mature, native SLT-1A polypeptide sequence, and a resulting catalytic residue disruption ( see also SEQ ID NOs: 23, 29, 40, 41, 42, and 43). Table 9.
  • All of the Shiga toxin effector polypeptides represented by SEQ ID NOs: 23, 29, 40, 42, and 43 comprise disruptions of a predicted endogenous CD4+ T-cell epitope(s).
  • the exemplary Shiga toxin effector polypeptides with embedded T-cell epitopes which do not disrupt any B-cell epitope region shown in Table 8 at least eight of them disrupt a catalytic amino acid residue of the Shiga toxin effector region (see SEQ ID NOs: 23, 25, 29, 31 35, and 37).
  • multiple immunogenic T-cell epitopes for MHC class I presentation are embedded and/or inserted within the same Shiga toxin-derived polypeptides or diphtheria toxin-derived polypeptides for use in the targeted delivery of a plurality of T-cell epitopes simultaneously, such as, e.g ., disrupting a B-cell epitope region with a first embedded T-cell epitope and disrupting a toxin catalytic function with a second embedded T-cell epitope.
  • a single embedded T-cell epitope can simultaneously disrupt both a B-cell epitope region and a toxin catalytic function.
  • the enzymatic and cytotoxic activities of the exemplary cytotoxic, cell-targeted proteins of the invention were compared to the parental Shiga toxin effector polypeptide alone (no cell-targeting binding region) and a "WT" cytotoxic protein comprising the same cell-targeting domain (e.g. binding region comprising an immunoglobulin-type binding region capable of binding an extracellular target biomolecule with high affinity) but with a wild-type Shiga toxin effector region.
  • the ribosome inactivation capabilities of cytotoxic proteins comprising embedded or inserted T-cell epitopes were determined using a cell-free, in vitro protein translation assay using the TNT® Quick Coupled Transcription/Translation kit (L1170 Promega Madison, WI, U.S.).
  • the kit includes Luciferase T7 Control DNA (L4821 Promega Madison, WI, U.S.) and TNT® Quick Master Mix.
  • the ribosome activity reaction was prepared according to manufacturer's instructions.
  • a series of 10-fold dilutions of the protein to be tested comprising either a mutated Shiga toxin effector polypeptide region or WT region, was prepared in an appropriate buffer and a series of identical TNT reaction mixture components were created for each dilution.
  • Each sample in the dilution series was combined with each of the TNT reaction mixtures along with the Luciferase T7 Control DNA.
  • the test samples were incubated for 1.5 hours at 30 degrees Celsius (°C). After the incubation, Luciferase Assay Reagent (E1483 Promega, Madison, WI, U.S.) was added to all test samples and the amount of luciferase protein translation was measured by luminescence according to manufacturer's instructions.
  • IC 50 values were calculated for each de-immunized protein comprising a B cell epitope replacement/disruption Shiga toxin effector polypeptide region and a control protein comprising a wild-type Shiga toxin effector region.
  • the exemplary Shiga toxin effector polypeptide regions of the invention exhibited ribosome inhibition comparable to a wild-type Shiga toxin effector polypeptide as indicated in Table 10. As reported in Table 10, any construct comprising a Shiga toxin effector polypeptide of the invention which exhibited an IC 50 within 10-fold of the positive control construct comprising a wild-type Shiga toxin effector region was considered to exhibit ribosome inhibition activity comparable to wild-type. Table 10.
  • cytotoxicity by exemplary Shiga toxin effector polypeptides of the invention after T-cell epitope embedding/insertion was determined by a cell-kill assay in the context of the Shiga toxin effector polypeptide as a component of a cytotoxic protein.
  • the cytotoxicity levels of proteins comprising Shiga toxin effector polypeptides, comprising an embedded or inserted T-cell epitope, were determined using extracellular target expressing cells as compared to cells that do not express a target biomolecule of the cytotoxic protein's binding region.
  • Cells were plated (2 x 10 3 cells per well for adherent cells, plated the day prior to protein addition or 7.5 x 10 3 cells per well for suspension cells, plated the same day as protein addition) in 20 ⁇ L cell culture medium in 384 well plates.
  • a series of 10-fold dilutions of each protein comprising a mutated Shiga toxin effector polypeptide region to be tested was prepared in an appropriate buffer, and then 5 ⁇ L of the dilutions or buffer control were added to the cells. Control wells containing only media were used for baseline correction.
  • the cell samples were incubated with the proteins or just buffer for 3 days at 37°C and in an atmosphere of 5% carbon dioxide (CO 2 ).
  • the total cell survival or percent viability was determined using a luminescent readout using the CellTiter-Glo® Luminescent Cell Viability Assay (G7573 Promega Madison, WI, U.S.) according to the manufacturer's instructions.
  • the Percent Viability of experimental wells was calculated using the following equation: (Test RLU - Average Media RLU) / (Average Cells RLU - Average Media RLU) * 100.
  • Log polypeptide concentration versus Percent Viability was plotted in Prism (GraphPad Prism, San Diego, CA, U.S.) and log (inhibitor) versus response (3 parameter) analysis was used to determine the half-maximal cytotoxic concentration (CD 50 ) value for the tested proteins.
  • the CD 50 was calculated for each protein comprising an exemplary Shiga toxin effector polypeptide of the invention in Table 10, positive-control cytotoxic protein comprising a wild-type Shiga toxin effector region, and the SLT-1 A subunit alone (no targeting domain).
  • the same protein constructs comprising exemplary Shiga toxin effector polypeptides of the invention exhibited specific cytotoxicity to biomolecular-target-expressing cells as compared to biomolecular-target-negative cells (i.e. cells which did not express, at a cellular surface, the biomolecular target of the cell-target binding region of the protein construct).
  • all the proteins comprising the exemplary Shiga toxin effector polypeptides in Table 10 were cytotoxic proteins exhibiting Shiga toxin effector functions comparable to wild-type, and each cytotoxic protein comprised a disruption in one or more predicted, B-cell epitope regions.
  • T-cell epitope embedded, diphtheria toxin effector polypeptides were compared to diphtheria toxin effector polypeptides comprising only wild-type amino acid sequences, referred to herein as "wild-type” or "WT.” Both T-cell epitope embedded, diphtheria toxin effector polypeptide variants retained ribosome inactivation activity.
  • diphtheria toxin effector polypeptide variants with embedded T-cell epitopes were tested using a ribosome inhibition assay and a wild-type diphtheria toxin effector polypeptide as a positive control.
  • the ribosome inactivation capabilities of these toxin effector polypeptides was determined using a cell-free, in vitro protein translation assay using the TNT® Quick Coupled Transcription/Translation kit (L1170 Promega Madison, WI, U.S.) as described above unless otherwise noted.
  • diphtheria toxin effector polypeptides were cleaved in vitro with furin (New England Biolabs, Ipswich, MA, U.S.) under standard conditions. Then the cleaved proteins were diluted in buffer to make a series of dilutions for each sample. Each dilution in each series was combined with each of the TNT reaction mixtures along with the Luciferase T7 Control DNA and tested for ribosome inactivation activity as described above.
  • the IC 50 was calculated, as described above, for the diphtheria toxin effector polypeptides.
  • Figure 2 and Table 11 show the results of this in vitro assay for retention of diphtheria ribotoxic toxin effector function by exemplary, T-cell embedded, diphtheria toxin effector polypeptides of the invention.
  • the activity of the T-cell embedded, diphtheria toxin effector polypeptides was comparable to the wild-type positive control because the IC 50 values were within ten-fold of the wild-type diphtheria toxin effector polypeptide control ( Figure 2 ; Table 11). Table 11.
  • the antigenicity or immunogenicity levels of Shiga toxin effector polypeptides was tested both in silico and by Western blotting using pre-formed antibodies which recognize wild-type Shiga toxin effector polypeptides.
  • cytotoxic proteins comprising a Shiga toxin effector polypeptide comprising either only a wild-type Shiga toxin sequence or one of various modified Shiga toxin sequences comprising a B-cell epitope region disruption via replacement with a T-cell epitope (SEQ ID NO: 11-19). These cytotoxic proteins were loaded in equal amounts to replicate, 4-20% sodium dodecyl sulfate (SDS), polyacrylamide gels (Lonza, Basel, CH) and electrophoresed under denaturing conditions.
  • SDS sodium dodecyl sulfate
  • Polyacrylamide gels Lionza, Basel, CH
  • the resulting gels were either analyzed by Coomassie staining or transferred to polyvinyl difluoride (PVDF) membranes using the iBlot® (Life Technologies, Carlsbad, CA, U.S.) system according to manufacturer's instructions.
  • PVDF polyvinyl difluoride
  • RGIDPEEGRFNN is located at amino acids 55-66 in SLT-1A and StxA, spanning a predicted B cell epitope
  • peptide sequence HGQDSVRVGR is located at 214-223 in SLT-1A and StxA, spanning a predicted B-cell epitope.
  • Disruptions in predicted CD4+ T-cell epitope regions are tested for reductions in CD4+ T-cell immunogenicity using assays of human CD4+ T-cell proliferation in the presence of exogenously administered polypeptides and assays of human CD4+ dendritic T-cell stimulation in the presence of human monocytes treated with administered polypeptides.
  • T-cell proliferation assays known to skilled worker are used to test the effectiveness of CD4+ T-cell epitope de-immunization in exemplary toxin effector polypeptides comprising T-cell epitopes embedded or inserted into predicted CD4+ T-cell epitopes.
  • the T-cell proliferation assay of this example involves the labeling of CD4+ T-cells and then measuring changes in proliferation using flow cytometric methods in response to the administration of different peptides derived from either a polypeptide de-immunized using the methods of embedding or inserting a heterologous CD8+ T-cell epitope (e.g., SEQ ID NOs: 11-43) or a reference polypeptide that does not have any heterologous T-cell epitope associated with it.
  • a heterologous CD8+ T-cell epitope e.g., SEQ ID NOs: 11-43
  • a reference polypeptide that does not have any heterologous T-cell epitope associated with it.
  • the CD8+ T-cell depleted, PBMCs that proliferate in response to an administered peptide will show a reduction in CFSE fluorescence intensity as measured directly by flow cytometry.
  • the Percentage Stimulation above background is determined for each stimulated sample, through comparison with results from an unstimulated sample, such as by ranking with regard to fluorescent signal, as negative, dim, or high.
  • Counts for the CD4+ CFSE T-cell dim population in each sample are expressed as a proportion of the total CD4+ T-cell population.
  • the replicate values are used to calculate Percentage Stimulation above Background (proportion of CD4+ T-cell CFSE dim cells with antigen stimulation, minus proportion of CD4+ T-cell CFSE dim cells without antigen stimulation).
  • the mean and standard error of the mean are calculated from the replicate values. A result is considered "positive” if the Percentage Stimulation above background is greater than 0.5% and also greater than twice the standard error above background.
  • a Response Index is calculated. This index is based on multiplying the magnitude of response (Percentage Stimulation above background) for each peptide by the number of responding donors (Percentage Antigenicity) for each peptide.
  • the relative CD4+ T-cell immunogenicity of exemplary, full-length polypeptides of the invention is determined using the following dendritic cell (DC) T-cell proliferation assay.
  • This DC T-cell assay measures CD4+ T-cell responses to exogenously administered polypeptides or proteins.
  • the DC T-cell assay is performed using ProImmune's DC-T assay service to determine the relative levels of CD4 + T-cell driven immunogenicity between polypeptides, proteins, and cell-targeted molecules of the invention as compared to the starting parental polypeptides, proteins, or cell-targeted molecules which lack the addition of any heterologous T-cell epitope.
  • the DC T-cell assay of this example involves testing human dendritic cells for antigen presentation of peptides derived from the administered polypeptide, protein, or cell-targeted molecule samples.
  • healthy human donor tissues are used to isolate typed samples based on high-resolution MHC Class II tissue-typing.
  • a cohort of 20, 40 or 50 donors is used.
  • monocytes obtained from human donor PBMCs are cultured in a defined medium to generate immature dendritic cells.
  • the immature dendritic cells are stimulate with a well-defined control antigen and induced into a more mature phenotype by further culture in a defined medium.
  • CD8+ T-cell depleted donor PBMCs from the same human donor sample is labeled with CFSE.
  • each dendritic cell culture series also includes a set of untreated dendritic cells.
  • the assay incorporates two well-defined reference antigens, each comprising a full-length protein.
  • the frequency of donor cell responses is analyzed across the study cohort. Positive responses in the assay are considered indicative of a potential in vivo CD4+ T-cell response.
  • a positive response measured as a percentage of stimulation above background, is defined as percentages greater than 0.5 percent % in 2 or more independent donor samples.
  • the strength of positive donor cell responses is determined by taking the mean percentage stimulation above background obtained across accepted donors for each sample.
  • a Response Index is calculated by multiplying the value of the strength of response by the frequency of the donors responding to determine levels of CD4+ T-cell immunogenicity for each sample.
  • a Response index representing the relative CD4+ T-cell immunogenicity is determined by comparing the results from two samples, one comprising a CD8+ T cell epitope embedded in a predicted CD4+ T-cell epitope region and a second variant which lacks any disruption to the same predicted CD4+ T-cell region to determine if the disruption reduces the CD4+ T-cell response of human donor cells.
  • mice are intravenously administered cytotoxic proteins or polypeptides comprising either wild-type or de-immunized forms of the Shiga toxin effector polypeptide component 3 times per week for two weeks or more.
  • Blood samples are taken from the injected mice and tested by enzyme-linked immunosorbent assay (ELISA) for reactivity to the cytotoxic proteins and/or the Shiga toxin effector polypeptide.
  • ELISA enzyme-linked immunosorbent assay
  • Reduced immunogenic responses will be elicited in mice injected with the de-immunized Shiga toxin effector polypeptide, or compositions comprising the same, as compared to mice injected only with the wild-type form of the Shiga toxin effector polypeptide, or composition comprising the same.
  • the relatively reduced immunogenic response will indicate that the de-immunized Shiga toxin effector polypeptides are de-immunized with regard to having reduced immunogenic potential after administration to a mammal and allowing time for the mammal's immune system to respond.
  • diphtheria toxin effector polypeptides of the invention are tested for de-immunization using the methods of this example to verify the disruption of one or more B-cell epitope regions in each diphtheria toxin effector polypeptides comprising an embedded or inserted T-cell epitope.
  • exemplary cell-targeted proteins of the invention which each comprise an exemplary Shiga toxin effector polypeptide of the invention, to deliver T-cell epitopes to the MHC class I pathway of target cells for presentation to the target cell surface was investigated.
  • cell-targeted proteins comprising diphtheria toxin effector polypeptides of the invention (e.g. SEQ ID NOs: 46-48) are tested using the methods of this example to verify their ability to deliver embedded T-cell epitopes to the MHC class I presentation system.
  • a cell-targeted protein of the invention comprises both a Shiga toxin effector polypeptide of the invention and a cell-targeting binding region capable of exhibiting high-affinity binding to an extracellular target biomolecule physically-coupled to the surface of a specific cell type(s).
  • the cell-targeted proteins of the invention are capable of selectively targeting cells expressing the target biomolecule of their cell-targeting binding region and internalizing into these target cells.
  • a flow cytometry method was used to demonstrate delivery and extracellular display of a T-cell epitope (inserted or embedded in a Shiga toxin effector region) in complex with MHC Class I molecules on the surfaces of target cells.
  • This flow cytometry method utilizes soluble human T-cell receptor (TCR) multimer reagents (Soluble T-Cell Antigen Receptor STARTM Multimer, Altor Bioscience Corp., Miramar, FL, U.S.), each with high-affinity binding to a different epitope-human HLA complex.
  • TCR soluble human T-cell receptor
  • Each STARTM TCR multimer reagent is derived from a specific T-cell receptor and allows detection of a specific peptide-MHC complex based on the ability of the chosen TCR to recognize a specific peptide presented in the context of a particular MHC class I molecule.
  • These TCR multimers are composed of recombinant human TCRs which have been biotinylated and multimerized with streptavidin. The TCR multimers are labeled with phycoerythrin (PE).
  • the TCR CMV-pp65-PE STARTM multimer reagent (Altor Bioscience Corp., Miramar, FL, U.S.) was used in this Example.
  • MHC class I pathway presentation of the CMV C2 peptide (NLVPMVATV) by human cells expressing the HLA-A2 can be detected with the TCR CMV-pp65-PE STARTM multimer reagent which exhibits high affinity recognition of the CMV-pp65 epitope-peptide (residues 495-503, NLVPMVATV) complexed to human HLA-A2 and is labeled with PE.
  • the target cells used in this Example were immortalized human cancer cells available from the ATCC (Manassas VA, U.S.). Using standard flow cytometry methods known in the art, the target cells were confirmed to express on their cell surfaces both the HLA-A2 MHC-Class I molecule and the extracellular target biomolecule of the proteins used in this Example.
  • the target cells were treated with the exemplary cell-targeted proteins of the invention, each comprising different Shiga toxin effector polypeptides comprising a T-cell epitope embedded into a predicted B-cell epitope region.
  • One of each of the exemplary cell-targeted proteins of the invention tested in this Example comprised one of the following Shiga toxin effector polypeptides: 43-51-C2 (SEQ ID NO:13), 53-61-C2(SEQ ID NO:17), and 104-112-C2(SEQ ID NO:18).
  • Sets of target cells were treated by exogenous administration of the different exemplary cell-targeted proteins of the invention at concentrations similar to those used by others taking into account cell-type specific sensitivities to Shiga toxins (see e.g. Noakes K et al., FEBS Lett 453: 95-9 (1999 )).
  • the treated cells were then incubated for six hours in standard conditions, including at 37°C and an atmosphere with 5% carbon dioxide, to allow for intoxication mediated by a Shiga toxin effector region. Then the cells were washed with cell culture medium, re-suspended in fresh cell culture medium, and incubated for 20 hours prior to staining with the TCR CMV-pp65-PE STARTM multimer reagent.
  • NLVPMVATV exogenously administered CMV C2 peptide
  • PLE Peptide Loading Enhancer
  • Cells displaying the appropriate MHC class I haplotype can be forced to load the appropriate exogenously applied peptide from an extracellular space (i.e. in the absence of cellular internalization of the applied peptide) or in the presence of PLE, which is a mixture of B2-microglobulin and other components.
  • the results of the flow cytometric analysis of the sets of differently treated cells are shown in Figure 5 and Table 14.
  • the untreated control was used to identify the positive and negative cell populations by employing a gate which results in less than 1% of cells from the untreated control in the "positive" gate (representing background signal). The same gate was then applied to the other samples to characterize the positive population for each sample.
  • the flow cytometry histograms are given with the counts (number of cells) on the Y-axis and the relative fluorescent units (RFU) on the X-axis (log scale). The grey line in all histograms shows the profile of the untreated cells and the black line shows the profile of treated cells according to the treatment indicated.
  • Table 14 the percentage of cells in a treatment set which stained positive for the C2-epitope-peptide-HLA-A2 complex is given. Positive cells in this assay were cells which were bound by the TCR-CMV-pp65-PE STAR reagent and counted in the positive gate described above. Table 14 also shows for each set the corresponding indexed, mean, fluorescent intensity ("iMFI," the fluorescence of the positive population multiplied by the percent positive) in RFU. Table 14.
  • Flow Cytometry Results for Exemplary Cell-targeted proteins of the invention Peptide-epitope C2-MHC class I complexes detected on the surfaces of intoxicated, target cells TCR CMV-pp65-PE Flow Cytometry Target cell treatment: exogenously administered molecule(s) Percentage of Positive Cells iMFI (RFU) Untreated 0.96% 20 Cell-targeted protein with Shiga toxin effector region 43-51-C2 7.6% 113 Cell-targeted protein with Shiga toxin effector region 53-61-C2 4.5% 64 Cell-targeted protein with Shiga toxin effector region 104-112-C2 6.7% 89 C2 peptide only 0.95% 19 C2 peptide and PLE 36.7% 728
  • the positive control "C2 peptide and PLE" population contained 36.7% positive cells; however, the peptide can only be loaded onto the surface from an extracellular space ("exogenously") and in the presence of PLE as shown by comparing with the "C2 peptide only” results which had a similar background staining level (0.95%) as the untreated control.
  • standard assays known in the art are used to investigate the functional consequences of target cells' MHC class I presentation of T-cell epitopes delivered by exemplary cell-targeted proteins of the invention.
  • the functional consequences to investigate include CTL activation, CTL mediated target cell killing, and CTL cytokine release by CTLs.
  • CTL response assays e.g . CytoTox96® non-radioactive assays (Promega, Madison, WI, U.S.), Granzyme B ELISpot assays (Mabtech, Inc., Cincinnati, OH, U.S.), caspase activity assays, and LAMP-1 translocation flow cytometric assays.
  • CTL response assays e.g . CytoTox96® non-radioactive assays (Promega, Madison, WI, U.S.), Granzyme B ELISpot assays (Mabtech, Inc., Cincinnati, OH, U.S.), caspase activity assays, and LAMP-1 translocation flow cytometric assays.
  • CTL response assays e.g . CytoTox96® non-radioactive assays (Promega, Madison, WI, U.S.), Granzyme B ELISpot assays (Mabtech, Inc., Cincinnati, OH, U.S.
  • a T-cell hyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga toxin effector region is derived from the A subunit of Shiga-like Toxin 1 (SLT-1A) as described above.
  • An immunoglobulin-type binding region ⁇ CD20-antigen is derived from an immunoglobulin-type domain recognizing human CD20 (see e.g.
  • CD20 is expressed on multiple cancer cell types, such as, e.g., B-cell lymphoma cells, hairy cell leukemia cells, B-cell chronic lymphocytic leukemia cells, and melanoma cells.
  • CD20 is an attractive target for therapeutics to treat certain autoimmune diseases, disorders, and conditions involving overactive B-cells.
  • the immunoglobulin-type binding region ⁇ CD20 and Shiga toxin effector region are linked together.
  • a fusion protein is produced by expressing a polynucleotide encoding the ⁇ CD20-antigen-binding protein SLT-1A:: ⁇ CD20 (see, e.g., SEQ ID NOs: 49, 50, and 51).
  • Expression of the SLT-1A:: ⁇ CD20 cytotoxic protein is accomplished using either bacterial and/or cell-free, protein translation systems as described in the previous examples.
  • the binding characteristics, the maximum specific binding (B max ) and equilibrium binding constants (K D ), of the cytotoxic protein of this example for CD20+ cells and CD20- cells is determined by fluorescence-based, flow-cytometry.
  • the B max for SLT-1A: : ⁇ CD20 to CD20+ cells is measured to be approximately 50,000-200,000 MFI with a K D within the range of 0.01-100 nM, whereas there is no significant binding to CD20- cells in this assay.
  • the ribosome inactivation abilities of the SLT-1A:: ⁇ CD20 cytotoxic protein is determined in a cell-free, in vitro protein translation as described above in the previous examples.
  • the inhibitory effect of the cytotoxic protein of this example on cell-free protein synthesis is significant.
  • the IC 50 of SLT-1A:: ⁇ CD20 on protein synthesis in this cell-free assay is approximately 0.1-100 pM.
  • the cytotoxicity characteristics of SLT-10A:: ⁇ CD20 are determined by the general cell-kill assay as described above in the previous examples using CD20+ cells.
  • the selective cytotoxicity characteristics of SLT-1A:: ⁇ CD20 are determined by the same general cell-kill assay using CD20- cells as a comparison to the CD20+ cells.
  • the CD 50 of the cytotoxic protein of this example is approximately 0.01-100 nM for CD20+ cells depending on the cell line.
  • the CD 50 of the cytotoxic protein is approximately 10-10,000 fold greater (less cytotoxic) for cells not expressing CD20 on a cellular surface as compared to cells which do express CD20 on a cellular surface.
  • the cytotoxicity of SLT-1A: : ⁇ CD20 is investigated for both direct cytotoxicity and indirect cytotoxicity by T-cell epitope delivery and presentation leading to CTL-mediated cytotoxicity.
  • mice Animal models are used to determine the in vivo effects of the cytotoxic protein SLT-1A:: ⁇ CD20 on neoplastic cells.
  • Various mice strains are used to test the effect of the cytotoxic protein after intravenous administration on xenograft tumors in mice resulting from the injection into those mice of human neoplastic cells which express CD20 on their cell surfaces.
  • Cell killing is investigated for both direct cytotoxicity and indirect cytotoxicity by T-cell epitope delivery and presentation leading to CTL-mediated cytotoxicity.
  • the CD8+ T-cell hyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga toxin effector region is derived from the A subunit of Shiga-like Toxin 1 (SLT-1A) as described above.
  • the immunoglobulin-type binding region is ⁇ HER2 V H H derived from a single-domain variable region of the camelid antibody (V H H) protein 5F7, as described in U.S. Patent Application Publication 2011/0059090 .
  • the immunoglobulin-type binding region and Shiga toxin effector region are linked together to form a fused protein (see, e.g., SEQ ID NOs: 52, 53, and 54).
  • a polynucleotide encoding the ⁇ HER2-V H H variable region derived from protein 5F7 may be cloned in frame with a polynucleotide encoding a linker known in the art and in frame with a polynucleotide encoding the Shiga toxin effector region comprising amino acids of SEQ ID NOs:11-43.
  • Variants of " ⁇ HER2-V H H fused with SLT-1A" cytotoxic proteins are created such that the binding region is optionally located adjacent to the amino-terminus of the Shiga toxin effector region and optionally comprises a carboxy-terminal endoplasmic reticulum signal motif of the KDEL family.
  • Expression of the " ⁇ HER2-V H H fused with SLT-1A" cytotoxic protein variants is accomplished using either bacterial and/or cell-free, protein translation systems as described in the previous examples.
  • the binding characteristics of the cytotoxic protein of this example for HER2+ cells and HER2- cells is determined by a fluorescence-based, flow-cytometry.
  • the B max for " ⁇ HER2-V H H fused with SLT-1A" variants to HER2+ cells is measured to be approximately 50,000-200,000 MFI with a K D within the range of 0.01-100 nM, whereas there is no significant binding to HER2- cells in this assay.
  • the ribosome inactivation abilities of the " ⁇ HER2-V H H fused with SLT-1A" cytotoxic proteins are determined in a cell-free, in vitro protein translation as described above in the previous examples.
  • the inhibitory effect of the cytotoxic protein of this example on cell-free protein synthesis is significant.
  • the IC 50 of " ⁇ HER2-V H H fused with SLT-1A" variants on protein synthesis in this cell-free assay is approximately 0.1-100 pM.
  • the cytotoxicity characteristics of " ⁇ HER2-V H H fused with SLT-1A" variants are determined by the general cell-kill assay as described above in the previous examples using HER2+ cells.
  • the selective cytotoxicity characteristics of " ⁇ HER2-V H H fused with SLT-1A” are determined by the same general cell-kill assay using HER2- cells as a comparison to the HER2+ cells.
  • the CD 50 of the cytotoxic protein of this example is approximately 0.01-100 nM for HER2+ cells depending on the cell line.
  • the CD 50 of the cytotoxic protein is approximately 10-10,000 fold greater (less cytotoxic) for cells not expressing HER2 on a cellular surface as compared to cells which do express HER2 on a cellular surface.
  • the cytotoxicity of ⁇ HER2-V H H fused with SLT-1A is investigated for both direct cytotoxicity and indirect cytotoxicity by T-cell epitope delivery and presentation leading to CTL-mediated cytotoxicity.
  • mice Animal models are used to determine the in vivo effects of the cytotoxic protein ⁇ HER2-V H H fused with SLT-1A on neoplastic cells.
  • Various mice strains are used to test the effect of the cytotoxic protein after intravenous administration on xenograft tumors in mice resulting from the injection into those mice of human neoplastic cells which express HER2 on their cell surfaces.
  • Cell killing is investigated for both direct cytotoxicity and indirect cytotoxicity by T-cell epitope delivery and presentation leading to CTL-mediated cytotoxicity.
  • the CD8+ T-cell hyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga toxin effector region is a de-immunized Shiga toxin effector polypeptide derived from the A subunit of Shiga-like Toxin 1 (SLT-1A) as described above.
  • SLT-1A Shiga-like Toxin 1
  • the cytotoxicity characteristics of SLT-1A:: ⁇ CCR5::KDEL are determined by the general cell-kill assay as described above in the previous examples using CCR5+ cells.
  • the selective cytotoxicity characteristics of SLT-1A:: ⁇ CCR5::KDEL are determined by the same general cell-kill assay using CCR5- cells as a comparison to the CCR5+ cells.
  • the CD 50 of the cytotoxic protein of this example is approximately 0.01-100 nM for CCR5+ cells depending on the cell line.
  • the CD 50 of the cytotoxic protein is approximately 10-10,000 fold greater (less cytotoxic) for cells not expressing CCR5 on a cellular surface as compared to cells which do express CCR5 on a cellular surface.
  • the cytotoxicity of SLT-1A:: ⁇ CCR5::KDEL is investigated for both direct cytotoxicity and indirect cytotoxicity by T-cell epitope delivery and presentation leading to CTL-mediated cytotoxicity.
  • SLT-1A:: ⁇ CCR5::KDEL The use of SLT-1A:: ⁇ CCR5::KDEL to block HIV infection is tested by giving an acute dose of SLT-1A:: ⁇ CCR5::KDEL to non-human primates in order to severely deplete circulating T-cells upon exposure to a simian immunodeficiency virus (SIV) (see Sellier P et al., PLoS One 5: e10570 (2010 )).
  • SIV simian immunodeficiency virus
  • the immunoglobulin-type binding region ⁇ Env and Shiga toxin effector region are linked together, and a carboxy-terminal KDEL is added to form a cytotoxic protein.
  • a fusion protein is produced by expressing a polynucleotide encoding the ⁇ Env-binding protein SLT-1A:: ⁇ Env::KDEL. Expression of the SLT-1A:: ⁇ Env::KDEL cytotoxic protein is accomplished using either bacterial and/or cell-free, protein translation systems as described in the previous examples.
  • the cytotoxicity characteristics of diphtheria toxin:: ⁇ CD20 are determined by the general cell-kill assay as described above in the previous examples using CD20+ cells.
  • the selective cytotoxicity characteristics of diphtheria toxin: : ⁇ CD20 are determined by the same general cell-kill assay using CD20- cells as a comparison to the CD20+ cells.
  • the CD 50 of the cytotoxic protein of this example is approximately 0.01-100 nM for CD20+ cells depending on the cell line.
  • the CD 50 of the cytotoxic protein is approximately 10-10,000 fold greater (less cytotoxic) for cells not expressing CD20 on a cellular surface as compared to cells which do express CD20 on a cellular surface.
  • the cytotoxicity of diphtheria toxin:: ⁇ CD20 is investigated for both direct cytotoxicity and indirect cytotoxicity by T-cell epitope delivery and presentation leading to CTL-mediated cytotoxicity.
  • mice Animal models are used to determine the in vivo effects of the cytotoxic protein diphtheria toxin: : ⁇ CD20 on neoplastic cells.
  • Various mice strains are used to test the effect of the cytotoxic protein after intravenous administration on xenograft tumors in mice resulting from the injection into those mice of human neoplastic cells which express CD20 on their cell surfaces.
  • Cell killing is investigated for both direct cytotoxicity and indirect cytotoxicity by T-cell epitope delivery and presentation leading to CTL-mediated cytotoxicity.
  • the CD8+ T-cell hyper-immunized and B-cell/CD4+ T-cell de-immunized diphtheria toxin effector region is derived from the A subunit of diphtheria toxin as described above.
  • the immunoglobulin-type binding region is ⁇ HER2 V H H derived from a single-domain variable region of the camelid antibody (V H H) protein 5F7, as described in U.S. Patent Application Publication 2011/0059090 .
  • the ribosome inactivation abilities of the " ⁇ HER2-V H H fused with diphtheria toxin" cytotoxic proteins is determined in a cell-free, in vitro protein translation as described above in the previous examples.
  • the inhibitory effect of the cytotoxic protein of this example on cell-free protein synthesis is significant.
  • the IC 50 of " ⁇ HER2-V H H fused with diphtheria toxin"on protein synthesis in this cell-free assay is approximately 0.1-100 pM.
  • the cytotoxicity characteristics of " ⁇ HER2-V H H fused with diphtheria toxin” are determined by the general cell-kill assay as described above in the previous examples using HER2+ cells.
  • the selective cytotoxicity characteristics of " ⁇ HER2-V H H fused with diphtheria toxin” are determined by the same general cell-kill assay using HER2- cells as a comparison to the HER2+ cells.
  • the CD 50 of the cytotoxic protein of this example is approximately 0.01-100 nM for HER2+ cells depending on the cell line.
  • the CD 50 of the cytotoxic protein is approximately 10-10,000 fold greater (less cytotoxic) for cells not expressing HER2 on a cellular surface as compared to cells which do express HER2 on a cellular surface.
  • the cytotoxicity of " ⁇ HER2-V H H fused with diphtheria toxin” is investigated for both direct cytotoxicity and indirect cytotoxicity by T-cell epitope delivery and presentation leading to CTL-mediated cytotoxicity.
  • Patent 7,910,104 B2 CD52 B-cell cancers such as lymphoma and leukemia, and B-cell related immune disorders, such as autoimmune disorders CD56 binding monoclonal antibody(s) Shin J et al., Hybridoma 18: 521-7 (1999 ) CD56 immune disorders and various cancers, such as lung cancer, Merkel cell carcinoma, myeloma CD79 binding monoclonal antibody(s) Zhang L et al., Ther Immunol 2: 191-202 (1995 ) CD79 B-cell cancers or B-cell related immune disorders CD248 binding scFv(s) Zhao A et al., J Immunol Methods 363: 221-32 (2011 ) CD248 various cancers, such as inhibiting angiogenesis EpCAM binding monoclonal antibody(s) Schanzer J et al., J Immunother 29: 477-88 (2006 ) EpCAM various cancers, such as ovarian cancer, malignant ascites, gastric cancer PSMA binding monoclonal
  • hemaglutinins and influenza matrix protein 2 viral infections Broadly neutralizing antibodies identified from patient samples Prabakaran et al., Front Microbiol 3: 277 (2012 ) Coronavirus surface antigens viral infections Broadly neutralizing antibodies identified from patient samples Prabakaran et al., Front Microbiol 3: 277 (2012 ) Henipaviruses surface antigens viral infections

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